Membrane Potential Imaging in the Nervous System: Methods and Applications

Membrane Potential Imaging in the Nervous System

Membrane Potential Imaging in the Nervous System Methods and Applications

Editors Marco Canepari INSERM U836, Grenoble Institute of Neuroscience Université Joseph Fourier Grenoble Cedex 9 France

Dejan Zecevic Yale University School of Medicine Department of Cellular and Molecular Physiology New Haven CT, USA

Editors Marco Canepari INSERM U836, Grenoble Institute of Neuroscience Université Joseph Fourier Grenoble Cedex 9 France [email protected]

Dejan Zecevic Yale University School of Medicine Department of Cellular and Molecular Physiology New Haven CT, USA [email protected]

Please note that additional material for this book can be downloaded from http://extras.springer.com ISBN 978-1-4419-6557-8 e-ISBN 978-1-4419-6558-5 DOI 10.1007/978-1-4419-6558-5 Springer New York Dordrecht Heidelberg London Library of Congress Control Number: 2010935804  Springer Science+Business Media, LLC 2010 All rights reserved. This work may not be translated or copied in whole or in part without the written permission of the publisher (Springer Science+Business Media, LLC, 233 Spring Street, New York, NY 10013, USA), except for brief excerpts in connection with reviews or scholarly analysis. Use in connection with any form of information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed is forbidden. The use in this publication of trade names, trademarks, service marks, and similar terms, even if they are not identified as such, is not to be taken as an expression of opinion as to whether or not they are subject to proprietary rights. Printed on acid-free paper Springer is part of Springer Science+Business Media (www.springer.com)

Preface

It has been said that the twenty-first century is the century of the photon. Optical techniques, especially those based on lasers, have reached an advanced state of complexity and have entered many disciplines of neurobiology. The optical readout of the nervous activity is an important part of the “photon revolution” bringing about predictions that the photon will progressively replace the electron for probing neuronal function. This volume entitled Membrane Potential Imaging in the Nervous. System; Methods and Applications describes optical techniques to monitor neuronal membrane potential signals. The imaging approach provides a method for monitoring electrical signaling in the brain with sub-micron and sub-millisecond resolution. Two main advantages over conventional electrical measurements make this experimental technique both unique and valuable: (a) the ability to record voltage transients simultaneously at hundreds or thousands of sites in the nervous system and (b) the ability to monitor signals from structures that are too small for electrode measurements (e.g., terminal dendritic branches, axons, and dendritic spines). This recording technique represents a complementary component in a rapidly developing avant-garde strategy to implement a relatively noninvasive all-optical investigation of living

neural circuits using light-activated ion channels and imaging technologies. The knowledge upon which voltage-imaging technique is based was generated over several decades and described in a wide variety of publications. The aim of the book is to provide a single comprehensive source of information on different types of voltage-imaging techniques, including overviews, methodo­logical details, examples of experimental measurements, and future developments. The book is structured in five sections, each containing several chapters written by experts and major contributors to particular topics. The volume starts with a historical perspective and fundamental principles of membrane potential imaging and continues to cover the measurement of membrane potential signals from dendrites and axons of individual neurons, measurements of the activity of many neurons with single cell resolution, monitoring of population signals from the nervous system, and concludes with the overview of new approaches to voltage imaging. The book is targeted at all scientists interested in this mature but also rapidly expanding imaging approach. Grenoble, France New Haven CT

Marco Canepari Dejan Zecevic

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Contents

Chapter 1: Historical Overview and General Methods of Membrane Potential Imaging Lawrence B. Cohen Chapter 2: Design and Use of Organic Voltage Sensitive Dyes Leslie M. Loew Chapter 3: Imaging Submillisecond Membrane Potential Changes from Individual Regions of Single Axons, Dendrites and Spines Marco Canepari, Marko Popovic, Kaspar Vogt, Knut Holthoff, Arthur Konnerth, Brian M. Salzberg, Amiram Grinvald, Srdjan D. Antic, and Dejan Zecevic

chapter 8: Monitoring Population Membrane Potential Signals During Functional Development of Neuronal Circuits in Vertebrate Embryos Yoko Momose-Sato, Katsushige Sato, and Kohtaro Kamino chapter 9: Imaging the Dynamics of Mammalian Neocortical Population Activity In Vivo Amiram Grinvald, David Omer, Shmuel Naaman, and Dahlia Sharon chapter 10: Imaging the Dynamics of Neocortical Population Activity in Behaving and Freely Moving Mammals Amiram Grinvald and Carl C.H. Petersen

Chapter 4: Combined Voltage and Calcium Imaging and Signal Calibration Marco Canepari, Peter Saggau, and Dejan Zecevic

chapter 11: Monitoring Membrane Voltage Using Two-Photon Excitation of Fluorescent Voltage-Sensitive Dyes

Chapter 5: Use of Fast-Responding VoltageSensitive Dyes for Large-Scale Recording of Neuronal Spiking Activity with Single-Cell Resolution

Jonathan A.N. Fisher and Brian M. Salzberg

William N. Frost, Jean Wang, Christopher J. Brandon, Caroline Moore-Kochlacs, Terrence J. Sejnowski, and Evan S. Hill

Gaddum Duemani Reddy and Peter Saggau

chapter 6: Monitoring Integrated Activity of Individual Neurons Using FRET-Based Voltage-Sensitive Dyes

chapter 12: Random-Access Multiphoton Microscopy for Fast Three-Dimensional Imaging

chapter 13: Second Harmonic Imaging of Membrane Potential Leslie M. Loew and Aaron Lewis

Kevin L Briggman, William B. Kristan, Jesús E. González, David Kleinfeld, and Roger Y. Tsien

chapter 14: Genetically Encoded Protein Sensors of Membrane Potential

chapter 7: Monitoring Population Membrane Potential Signals from Neocortex

Lei Jin, Hiroki Mutoh, Thomas Knopfel, Lawrence B. Cohen, Thom Hughes, Vincent A. Pieribone, Ehud Y. Isacoff, Brian M. Salzberg, and Bradley J. Baker

Xiaoying Huang, Weifeng Xu, Kentaroh Takagaki, and Jian-Young Wu

Index

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Contributors

Srdjan D. Antic Department of Neuroscience, UConn Health Center, Farmington, CT 06030, USA Bradley J. Baker Department of Cellular and Molecular Physiology, Yale Uniersity School of Medicine, New Haven, CT 06520, USA Christopher J. Brandon Department of Cell Biology and Anatomy, The Chicago Medical School, Rosalind Franklin University of Medicine and Science, North Chicago, IL 60064, USA Kevin L. Briggman Department of Biomedical Optics, Max Planck Institute for Medical Research, Jahnstrasse 2969120, Heidelberg, Germany Marco Canepari Division of Pharmacology and Neurobiology, Biozentrum-University of Basel, Klingelbergstrasse 50-70, CH-4056 Basel, Switzerland [email protected] Lawrence B. Cohen Department of Physiology, Yale University School of Medicine, 333 Cedar St. New Haven, CT 06510, USA [email protected] Jonathan A.N. Fisher The Rockefeller University, New York City, NY, USA William N. Frost Department of Cell Biology and Anatomy, The Chicago Medical School, Rosalind Franklin University of Medicine and Science, North Chicago, IL 60064, USA [email protected] Jesús E. González 6468 Wayfinders Court, Carlsbad, CA 92011, USA Amiram Grinvald Department of Neurobiology, Weizmann Institute of Science, Rehovot 76100, PO Box 26, Israel [email protected]

Evan S. Hill Department of Cell Biology and Anatomy, The Chicago Medical School, Rosalind Franklin University of Medicine and Science, North Chicago, IL 60064, USA Knut Holthoff Hans Berger Klinik für Neurologie, Friedrich-Schiller-Universität Jena, Jena, Germany Xiaoying Huang Department of Physiology and Biophysics, Georgetown University Medical Center, 3900 Reservoir Road MW, Washington, DC 20007, USA Thom Hughes Department of Cell Biology and Neuroscience, Montana State University, Bozeman, Montana 59717, USA Ehud Y. Isacoff Department of Molecular and Cell Biology, University of California, Berkley, CA 94720, USA Lei Jin Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, CT 06520, USA Kohtaro Kamino Tokyo Medical and Dental University/ Chiba-Kashiwa Rehabilitation College David Kleinfeld Department of Physics, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0374, USA Thomas Knopfel Laboratory for Neuronal Circuit Dynamics, Brain Science Institute, RIKEN, 2-1 Hirosawa, Wako-Shi, Saitama 351-0198, Japan Arthur Konnerth Center for Intergrated Protein Science and Institute of Neuroscience, Technical University Munich, Munich, Germany William B. Kristan Neurobiology Section, Division of Biological Sciences, University of California, San Diego 9500 Gilman Drive, La Jolla, CA 92093-0357, USA [email protected]

Aaron Lewis Department of Applied Physics, Hebrew University, Jerusalem, Israel [email protected] Leslie M. Loew Department of Cell Biology, R. D. Berlin Center for Cell Analysis and Modeling, University of Connecticut Health Center, Farmington, CT 06030-1507, USA [email protected] Yoko Momose-Sato Department of Health and Nutrition, College of Human Environmental Studies, Kanto Gakuin University [email protected] Caroline Moore-Kochlacs Howard Hughes Medical Institute, The Salk Institute for Biological Studies, La Jolla, CA 92037, USA Hiroki Mutoh Laboratory for Neuronal Circuit Dynamics, Brain Science Institute, RIKEN, 2-1 Hirosawa, Wako-Shi, Saitama 351-0198, Japan Shmuel Naaman Department of Neurobiology, Weizmann Institute of Science, Rehovot 76100, PO Box 26, Israel David Omer Department of Neurobiology, Weizmann Institute of Science, Rehovot 76100, PO Box 26, Israel Carl C.H. Petersen Laboratory of Sensory Processing, Brain Mind Institute, Faculty of Life Sciences, École Polytechnique Fédérale de Lausanne (EPFL), CH-1015 Lausanne, Switzerland [email protected] Vincent A. Pieribone Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, CT 06520, USA and John B. Pierce Laboratory, New Haven, CT 06520, USA Marko Popovic Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, CT 06520, USA

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Contributors

Gaddum Duemani Reddy Department of Neuroscience, Baylor College of Medicine, Houston, TX 77030, USA

and Howard Hughes Medical Institute, The Salk Institute for Biological Studies, La Jolla, CA 92037, USA

Peter Saggau Department of Neuroscience, Baylor College of Medicine, Houston, TX 77030, USA [email protected]

Dahlia Sharon Department of Neurobiology, Weizmann Institute of Science, Rehovot 76100, PO Box 26, Israel and Department of Psychology, Stanford University, Stanford, CA 94305, USA

Brian M. Salzberg Department of Neuroscience, University of Pennsylvania School of Medicine, 234 Stemmler Hall Philadelphia, PA 19104-6074, USA and Departments of Neurobiology and Physiology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania [email protected] Katsushige Sato Department of Health and Nutrition Sciences, Faculty of Human Health, Komazawa Women’s University Terrence J. Sejnowski Division of Biological Sciences, University of California San Diego, La Jolla, CA 92093, USA

Kentaroh Takagaki Department of Physiology and Biophysics, Georgetown University Medical Center, 3900 Reservoir Road MW, Washington, DC 20007, USA Roger Y. Tsien Department of Pharmacology, Howard Hughes Medical Institute, University of California, San Diego 310 George Palade Labs, 9500 Gilman Drive, La Jolla, CA 92093-0647, USA Kaspar Vogt Division of Pharmacology and Neurobiology, Biozentrum – University of Basel, Basel, Switzerland

Jean Wang Department of Cell Biology and Anatomy, The Chicago Medical School, Rosalind Franklin University of Medicine and Science, North Chicago, IL 60064, USA Jian-young Wu Department of Physiology and Biophysics, Georgetown University Medical Center, 3900 Reservoir Road MW, Washington, DC 20007, USA [email protected] Weifeng Xu Department of Physiology and Biophysics, Georgetown University Medical Center, 3900 Reservoir Road MW, Washington, DC 20007, USA Dejan Zecevic Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, CT 06520, USA [email protected]

1

Historical Overview and General Methods of Membrane Potential Imaging Lawrence B. Cohen

1.1  Motivation

1.2  History

Several factors make optical methods interesting to physiologists and neuroscientists. First, is the possibility of making simultaneous measurements from multiple sites. Because many different neurons in a nervous system are active during each behavior, it is obviously important to be able to monitor activity from many cells simultaneously. Similarly, different parts of a neuron may perform different functions and again it would be essential to make simultaneous observations over the whole structure of the neuron. Finally, many different brain regions are simultaneously active during behaviors, and recording of population signals from these regions would be useful. Second, optical measurement can be made with very high time resolution. Some voltage-sensitive dyes are known to respond to changes in membrane potential extremely rapidly, with time constants of less than 1 ms. Other indicators of activity, such as hemodynamic changes, monitored as intrinsic signals, can be much slower, with latencies and durations in the range of seconds, but even so these recordings reflect real-time measurements of events in the brain. Third, optical measurements are in some sense noninvasive; recordings can be made from cells and processes too small for electrode measurements and these include T-tubules, presynaptic terminals, and dendritic spines. On the other hand, optical measurements using extrinsic dyes can be invasive if they cause pharmacological or photodynamic effects. The degree of this kind of invasiveness varies from relatively innocuous to measurements where pharmacology and photodynamic damage are important factors, a subject discussed in detail in later chapters. The above three factors are clearly attractive because the use of voltage imaging has grown dramatically since its beginnings in the early 1970s. Nonetheless, it is important to note that optical methods cannot be used to record from deep in a tissue, an important capability which other imaging methods such as fMRI and 2-deoxyglucose do provide. Three kinds of optical signals have been extensively used to monitor activity: dye indicators of membrane potential or intracellular ion concentration and intrinsic signals. This chapter is a historical and technical introduction of voltage imaging.

1.2.1  Early Milestones in Imaging Activity The first optical signals detected during nerve activity were light scattering changes that accompany trains of stimuli to nerves (Hill and Keynes 1949; Hill 1950). During and following the stimuli there is first an increase in scattering followed by a slower and long-lasting decrease lasting many tens of seconds (Fig. 1.1A). On the top left is a photograph of Richard Keynes (together with an oscilloscope) taken at the time of those measurements; on the top right is a photograph of David Hill taken about 20 years later. In the bottom panel is the recording of light scattering from a whole crab leg nerve as a function of time during and following a train of 250 stimuli. The light scattering response is shown on two time bases. Starting at about the same time and continuing into the 1960s, scientists at the Institute of Cytology in Leningrad began measuring changes in the optical properties of dye stained nerves (Nasonov and Suzdal’skaia 1957; Vereninov et al. 1962; Levin et al. 1968). Again they used relatively long trains of stimuli and recorded signals with time courses of tens of seconds. The photograph in Fig. 1.1B includes two of the scientists: D. N. Nasonov (standing) and Dora Rosenthal (seated). On the right is a recording from Levin et al. (1968) illustrating the change in absorption of the dye Direct Turquoise from an isolated crab axon. With the introduction of the signal averager to biological research it became possible to measure signals that were much smaller. One could then look for signals that occurred in coincidence with the action potential. This led to the discovery of changes in light scattering and birefringence that accompany the action potentials in nerves and single axons (Cohen et al. 1968). Figure 1.2A shows the first measurement of the birefringence signal from a squid giant axon made at the Laboratory of the Marine Biological Association in 1967. The signal was really small; the fractional change, ΔI/I, was only 1 part in 100,000. And, 20,000 trials had to be averaged to achieve the signal-to-noise ratio shown in the figure. Brushing aside the tiny size of the signals, David Gilbert (Fig. 1.2B; personal communication) pointed out that optical signals might be used to follow activity in the nervous system. Soon thereafter Ichiji

Lawrence B. Cohen  •  Department of Physiology, Yale University School of Medicine, 333 Cedar St. New Haven, CT 06510, USA M. Canepari and D. Zecevic (eds.), Membrane Potential Imaging in the Nervous System: Methods and Applications, DOI 10.1007/978-1-4419-6558-5_1, © Springer Science+Business Media, LLC 2010

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Chapter 1    L.B. Cohen ●

FIGURE 1.1.  (A) Top. Photographs of Richard Keynes and David Hill. Bottom panel. A Carcinus maenas leg nerve was stimulated 250 times during the bar labeled stimulus. The stimulus resulted in an increase in scattering that lasted about 20 s. This was followed by a scattering decrease which persisted for several minutes. From Hill and Keynes (1949). (B) A photograph of D.N. Nasonov and Dora Rosenthal taken in 1956 together with a recording of the change in Direct Turquoise absorption as a result of stimulating a crab axon 50 times/s for 2 min (6,000 stimuli). The optical recording is from Levin et al. (1968).

Tasaki (Fig. 1.2B) and collaborators (Tasaki et al. 1968) discovered fast changes in the fluorescence of axons that were stained with dyes. Several years later changes in dye absorption (Ross et  al. 1974) and birefringence (Ross et al. 1977) were also found. Almost all of these dye signals were shown to depend on changes in membrane potential (Davila et al. 1974; Cohen et al. 1974; Salzberg

and Bezanilla 1983), a conclusion that is now widely accepted. However, there was early disagreement (Conti et  al. 1971) and Ichiji remained unconvinced. Ichiji (98) and Richard keynes (90) died during the preparation of this manuscript. In 1971, we began a deliberate search for larger optical signals by trying many dyes. After a few years we had obtained signals

Historical Overview and General Methods of Membrane Potential Imaging    Chapter 1 ●

3

et  al. 1975). More recently, Roger Tsien and collaborators have developed a large number of widely used ion indicator dyes based on the chemical structure of the chelator EGTA (e.g., Tsien 1989). Figure 1.4 illustrates two time-course comparisons between voltage, calcium, and intrinsic signals. The top pair compares voltage and calcium signals from the same location on a turtle olfactory bulb. The signals are in response to a shock to the olfactory nerve. The voltage-sensitive dye signal rises faster and falls faster than the calcium signal. Some of the slowness of the calcium signal is thought to result from the dye response itself and some from the time taken by the cells to return their calcium concentration to the original level. The bottom pair of traces compares a calcium signal and an intrinsic signal (Blasdel and Salama 1986; Grinvald et al. 1986) from the same glomerulus in a mouse olfactory bulb. Here the stimulus was an odorant presented to the nose. The calcium signal reaches a peak in less than 0.5 s, while the intrinsic signal does not reach its peak until about 5 s after the stimulus onset.

1.2.3  Multisite Measurements of Brain Activity The first multisite measurements of brain activity were made by Schuette et al. (1974) who used an image intensifier and camera tube to monitor changes in NADH fluorescence from the cortex during epileptic seizures. This was followed by methods using several individual silicon photodiodes (Salzberg et al. 1977), and later by a variety of photodiode arrays, camera tubes, CCD cameras, and CMOS cameras. FIGURE 1.2.  (A) The first recording of an optical signal (birefringence) from an individual axon on a time scale appropriate for measuring the time course of the signal during the action potential. Twenty thousand trials were averaged. In this and subsequent figures, the size of the vertical line represents the stated value of the fractional change in intensity ( I/I or F/F ). (L.B. Cohen and R.D. Keynes, unpublished result) (B) Photographs of David Gilbert and Ichiji Tasaki.

from 1,000 dyes, some that were about 100 times larger than the intrinsic signals described above. Hundred times larger does not mean that the signals are now large. Care is still needed to optimize the measurement of signal-to-noise ratio. Section 1.2 in this chapter, as well as other chapters in this book, provides information about the needed optimization. Because of this improvement in signal size, we were able to record optical signals from a smaller membrane area and with higher time resolution. The top panel in Fig. 1.3 illustrates a single trial absorption signal from a single action potential in a 50-mm diameter barnacle neuron made with the merocyanine dye whose structure is illustrated. The five adults on the right-hand side of the photograph at the bottom were in large part responsible for the dye screening. Other important contributors during those early years (not shown) were Vicencio Davila, Jeff Wang, Ravinder Gupta, and Alan Waggoner.

1.2.2  I on Sensitive Dyes and Their Comparison with Voltage-Sensitive Dyes The first attempts to use optical indicators of intracellular ion concentration were measurements of calcium with the dye murexide (Jobsis and O’Connor 1966). Ironically, those results are now thought to be an artifact (Maylie et  al. 1987). Later, Ashley and Ridgway (1970) introduced the use of the photoprotein aequorin. Following the suggestion of John Cooper (personal communication), the Arsenazo class of calcium dyes was introduced (Brown

1.3  Principles Even though the changes in membrane potential during action potentials and synaptic activity are small in mV, they are large in V/cm because the membrane is very thin. Thus, 100 mV across 3 nm is 300,000V/cm, a voltage gradient long known to be able to alter the spectral properties of merocyanine dyes (Labhart 1963; Bucher et  al. 1969). This direct effect of voltage on dye spectra, called electrochromism, is one mechanism thought to give rise to signals from organic voltage-sensitive dyes (Loew et  al. 1985). Other mechanisms with spectral supporting evidence are voltage-sensitive shifts in monomer–dimer equilibria (Waggoner and Grinvald 1977) and dye rotation in the electric field (Conti 1975; Fromherz et al. 1991). Because the electric field change is so large, it is perhaps not surprising that we were able to measure signals in ~500 of the ~2,000 dyes that were tested on squid giant axons. Action potential propagation and synaptic currents also give rise to extracellular and intracellular currents which means that there will also be voltage gradients in these locations. However, these extracellular currents flow over distances of mm (rather than nm), and thus the voltage gradients are smaller by six orders of magnitude. It is for this reason that the voltage-sensitive dye signals are presumed to arise solely from changes in dye molecules embedded in or directly adjacent to the membrane.

1.4  The Definition of a Voltage-sensitive Dye The voltage-sensitive dye signals described in this book are “fast” signals (Cohen and Salzberg 1978) that are presumed to arise from membrane-bound dye. Figure 1.5 illustrates the kind of result that is used to define a voltage-sensitive dye. In a giant axon from a

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Chapter 1    L.B. Cohen ●

FIGURE 1.3.  On the top is an absorption change measured during a single action potential from a cell body in a barnacle supraesophageal ganglion. The structure of the absorption dye, synthesized by Jeff Wang and Alan Waggoner, is illustrated at the top. In the photograph are, from the left, Naoko Kamino, Kyoko Kamino, Irit Grinvald, Eran Grinvald, Amiram Grinvald, Larry Cohen, Kohtaro Kamino, Kaeko Kamino, Brian Salzberg, and Bill Ross. The photograph is from 1976. The experiment is from Salzberg et al. (1977).

squid, these optical signals are fast, following membrane potential with a time constant of 1 kHz. While the need to provide a separate amplifier for each diode element limits the number of pixels in parallel readout systems, it contributes to the very large (105) dynamic range that can be achieved. The commercially available system NeuroPlex-III (464 pixels) is available from RedShirtImaging, LLC, Decatur, GA (www.redshirtimaging.com).

1.9.4  CCD Cameras By using a serial readout, the number of amplifiers is greatly reduced. In addition, it is easier to cool charge-coupled device (CCD) chips to reduce the dark noise. However, because of saturation, presently available CCD cameras are not optimal for the higher intensities available in some neurobiological experiments (Fig. 1.7). The high intensity limit of the CCD camera is set by the light intensity which fills the electron wells on the CCD chip. The well depth of commercially available CCD chips is less than 106 e–. This accounts for the bending over of the CCD camera performance at segment C in Fig. 1.7. A dynamic range of even 103 is not easily achieved with a CCD camera. A CCD camera will not be optimal for measurements of absorption or for fluorescence measurements on bulk-stained in vitro brain slices or intact brains (Fig. 1.7). The incident light intensity would have to be reduced with a consequent decrease in signal-to-noise ratio. On the other hand, CCD cameras are close to ideal for measurements from individual neurons stained with internally injected dyes.

1.9.5  CMOS Cameras SciMedia (Taito-ku, Tokyo) and RedShirtImaging now market CMOS cameras with a well depth of 106 e––108 e– that are close to ideal for the range of intensities between 105 and 108 photons/ms/ pixel. Table 1.2 compares a CCD camera, the photodiode array, and the CMOS cameras.

1.9.6  EM-CCD Cameras These cameras have on chip multiplication and should lead to better signal-to-noise performance at very low light levels. However, the multiplication process adds noise (a factor of 1.4) and some

existing chips are even noisier than expected from the factor of 1.4. If an ordinary CCD has a read noise of 10 e–, then an ideal EM-CCD camera will have a better signal-to-noise ratio only at light levels less than 100 photons/pixel/frame. The light level achieved in neurobiological and cardiac experiments is almost always greater than 1,000 photons/pixel/ms. Thus, CCD or CMOS cameras will have a better signal-to-noise ratio than an EM-CCD.

1.10  Organic Versus Genetically Encoded Voltage Sensors Organic voltage and calcium sensitive dyes stain all the cell types in the preparation. In some situations, this is a positive feature but in others it has negative consequences. In the vertebrate CNS, the uniform staining makes it painfully difficult to determine which cell types are responsible for which components of a population signal. Similarly in a two-photon measurement of calcium signals from cell bodies in the CNS, there is no easy way to determine which of the many cell types present are responding. In principle, a protein sensor of membrane potential or calcium would get around this problem. Once a promoter is identified that drives expression in a specific cell type, it would be possible to use that promoter to drive the expression of the protein sensor in that cell type in a transgenic mouse. Efforts are underway to develop useful protein sensors (see Chap. 14) Organic voltage-sensitive dyes were first introduced in the 1970s. Looking back it is clear that there has been remarkable progress in the development and utilization of these tools over the past 35 years. On the other hand, from the point of view of participants in these developments, progress has seemed to be painfully slow. We should take heart from the fact that the development of the microscope continues apace after more than 400 years of effort. Voltage-sensitive dye recording methods have already been applied to a wide variety of neurobiological problems that range from dendritic diversity in single neurons to activity maps that cover large areas of mammalian cortex. It seems likely that new applications and tools will continue to be developed.

Acknowledgments The author is indebted to his collaborators Vicencio Davila, Amiram Grinvald, Kohtaro Kamino, Ying-wan Lam, Leslie Loew, Bill Ross, Brian Salzberg, Alan Waggoner, Matt Wachowiak, Jian-young Wu, and Michal Zochowski for numerous discussions about optical methods. The experiments carried out in my laboratory were supported by NIH grants.

TABLE 1.2.  Characteristics of Fast CCD, CMOS, and PDA Camera Systems (as Reported by the Manufacturer) Frame Rate (Hz) Full Frame RedShirtImaging NeuroCCD-SMQ RedShirtImaging NeuroCMOS-SMa SciMedia MiCAM Ultimab CMOS RedShirtImaging NeuroPDAa a

www.redshirtimaging.com www.scimedia.com

a 

b 

2,000 10,000 10,000 1,600

Well Size (×1,000e) 200 100,000 100,000 1,000,000

Read Noise (electrons)

   Back Illumination

Bits a–d

Pixels

20 100 300 –

Yes No No Yes

14 14 14 18

80 × 80 128 × 128 100 × 100 ~24 × 24

Historical Overview and General Methods of Membrane Potential Imaging    Chapter 1 ●

References Ashley CC, Ridgway EB (1970) On the relationship between membrane potential, calcium transient and tension in single barnacle muscle fibres. J Physiol 209:105–130. Baker B, Kosmidis E, Vucinic D, Falk CX, Cohen LB, Djurisic M, Zecevic D (2005) Imaging brain activity with voltage- and calcium-sensitive dyes. Cell Mol Neurobiol 25:245–282. Blasdel GG, Salama G (1986) Voltage-sensitive dyes reveal a modular organization in monkey striate cortex. Nature 321:579–585. Boyle MB, Cohen LB (1980) Birefringence signals that monitor membrane potential in cell bodies of molluscan neurons. Fed Proc 39:2130. Braddick HJJ (1960) Photoelectric photometry. Rep Prog Phys 23:154–175. Brown JE, Cohen LB, De Weer P, Pinto LH, Ross WN, Salzberg BM (1975) Rapid changes in intracellular free calcium concentration. Detection by metallochromic indicator dyes in squid giant axon. Biophys J 15:1155–1160. Bucher H, Wiegand J, Snavely BB, Beck KH, Kuhn H (1969) Electric field induced changes in the optical absorption of a merocyanine dye. Chem Phys Lett 3:508–511. Bullen A, Patel SS, Saggau P (1997) High-speed, random-access fluorescence microscopy: I. High resolution optical recording with voltage-sensitive dyes and ion indicators. Biophys J 73:477–491. Cohen LB, Salzberg BM (1978) Optical measurement of membrane potential. Rev Physiol Biochem Pharmacol 83:35–88. Cohen LB, Keynes RD, Hille B (1968) Light scattering and birefringence changes during nerve activity. Nature 218:438–441. Cohen LB, Salzberg BM et  al (1974) Changes in axon fluorescence during activity: molecular probes of membrane potential. J Membr Biol 19:1–36. Conti F (1975) Fluorescent probes in nerve membranes. Annu Rev Biophys Bioeng 4:287–310. Conti F, Tasaki I, Wanke E (1971) Fluorescence signals in ANS-stained squid axons during voltage clamp. Biophys J 8:58–70. Davila HV, Cohen LB, Salzberg BM, Shrivastav BB (1974) Changes in ANS and TNS fluorescence in giant axons from Loligo. J Membr Biol 15:29–46. Fromherz P, Dambacher KH et al (1991) Fluorescent dyes as probes of voltage transients in neuron membranes: progress report. Ber Bunsenges Phys Chem 95:1333–1345. Garaschuk O, Milos RI, Grienberger C, Marandi N, Adelsberger H, Konnerth A (2006) Optical monitoring of brain function in  vivo: from neurons to networks. Pflugers Arch 453:385–396. Grinvald A, Lieke E, Frostig RD, Gilbert CD, Wiesel TN (1986) Functional architecture of cortex revealed by optical imaging of intrinsic signals. Nature 324:361–364. Gross E, Bedlack RS, Loew LM (1994) Dual-wavelength ratiometric fluorescence measurements of the membrane dipole potential. Biophys J 67:208–216. Gupta RK, Salzberg BM, Grinvald A, Cohen LB, Kamino K, Lesher S, Boyle MB, Waggoner AS, Wang CH (1981) Improvements in optical methods for measuring rapid changes in membrane potential. J Membr Biol 58:123–137. Hill DK (1950) The effect of stimulation on the opacitiy of a crustacean nerve trunk and its relation to fibre diameter. J Physiol 111:283–303. Hill DK, Keynes RD (1949) Opacity changes in stimulated nerve. J Physiol 108:278–281. Hirota A, Sato K, Momose-Sato Y, Sakai T, Kamino K (1995) A new simultaneous 1020-site optical recording system for monitoring neural activity using voltage-sensitive dyes. J Neurosci Meth 56:187–194. Inoue S (1986) Video microscopy. Plenum Press, New York. Jobsis FF, O’Connor MJ (1966) Calcium release and reabsorption in the sartorius muscle of the toad. Biochem Biophys Res Commum 25:246–252.

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Kleinfeld D, Delaney KR (1996) Distributed representation of vibrissa movement in the upper layers of somatosensory cortex revealed with voltage-sensitive dyes. J Comp Neurol 375:89–108. Labhart H (1963) Bestimmung von moleküleigenschaften aus elektrooptischen effekten. Tetrahedron 19(Suppl 2):223–241. Levin SV, Rosenthal DL, Komissarchik YY (1968) Structural changes in the axon membrane on excitation. Biofizika 13:180–182. Loew LM, Cohen LB, Salzberg BM, Obaid AL, Bezanilla F (1985) Charge-shift probes of membrane potential. Characterization of aminostyrylpyridinium dyes on the squid giant axon. Biophys J 47:71–77. London JA, Zecevic D, Cohen LB (1987) Simultaneous optical recording of activity from many neurons during feeding in Navanax. J Neurosci 7:649–661. Malmstadt HV, Enke CG, Crouch SR, Harlick G (1974) Electronic measurements for scientists. Benjamin, Menlo Park. Maylie J, Irving M, Sizto NL, Boyarsky G, Chandler WK (1987) Calcium signals recorded from cut frog twitch fibers containing tetramethylmurexide. J Gen Physiol 89:145–176. Nasonov DN, Suzdal’skaia IP (1957) Changes in the cytoplasm of myelinated nerve fibers during excitation. Fiziol Zh SSSR 43:664–672. Orbach HS, Cohen LB (1983) Optical monitoring of activity from many areas of the in  vitro and in  vivo salamander olfactory bulb: a new method for studying functional organization in the vertebrate central nervous system. J Neurosci 3:2251–2262. Orbach HS, Cohen LB, Grinvald A (1985) Optical mapping of electrical activity in rat somatosensory and visual cortex. J Neurosci 5:1886–1895. Ross WN, Salzberg BM, Cohen LB, Davila HV (1974) A large change in dye absorption during the action potential. Biophys 14:983–986. Ross WN, Salzberg BM et  al (1977) Changes in absorption, fluorescence, dichroism, and birefringence in stained giant axons: optical measurement of membrane potential. J Membr Biol 3:141–183. Salzberg BM, Bezanilla F (1983) An optical determination of the series resistance in Loligo. J Gen Physiol 82:807–817. Salzberg BM, Grinvald A, Cohen LB, Davila HV, Ross WN (1977) Optical recording of neuronal activity in an invertebrate central nervous system: simultaneous monitoring of several neurons. J Neurophysiol 40:1281–1291. Schuette WH, Whitehouse WC, Lewis DV, O’Connor M, VanBuren JM (1974) A television fluorimeter for monitoring oxidative metabolism in intact tissue. Med Instrum 8:331–333. Shaw R (1979) Photographic detectors. Appl Opt Optical Eng 7:121–154. Svoboda K, Denk W, Kleinfeld D, Tank DW (1997) In vivo dendritic calcium dynamics in neocortical pyramidal neurons. Nature 385:161–165. Tank D, Ahmed Z (1985) Multiple-site monitoring of activity in cultured neurons. Biophys J 47:476A. Tasaki I, Watanabe A, Sandlin R, Carnay L (1968) Changes in fluorescence, turbidity, and birefringence associated with nerve excitation. Proc Natl Acad Sci U S A 61:883–888. Tsien RY (1989) Fluorescent probes of cell signaling. Annu Rev Neurosci 12:227–253. Vereninov AA, Nikolsky NN, Rosenthal DL (1962) Neutral red sorption by the giant axon of Sepia at excitation. Tsitologiya 4:666–668. Wachowiak M, Cohen LB (2003) Correspondence between odorant-evoked patterns of receptor neuron input and intrinsic optical signals in the mouse olfactory bulb. J Neurophysiol 89:1623–1639. Waggoner AS, Grinvald A (1977) Mechanisms of rapid optical changes of potential sensitive dyes. Ann NY Acad Sci 303:217–241. Wu JY, Cohen LB (1993) Fast multisite optical measurement of membrane potential. In Fluorescent and Luminescent Probes for Biological Activity., WT Mason ed., Academic Press, London 389–404.

2

Design and Use of Organic Voltage Sensitive Dyes Leslie M. Loew

2.1  History and Introduction The pioneering work of Lawrence Cohen in the mid-1970s led to the establishment of optical methods as a way to measure the electrical activity of large populations of cells either through a microscope or in bulk suspension where traditional microelectrode methods are not applicable. Cohen’s laboratory used an extensive and comprehensive assay of commercially available dyes on the voltage-clamped squid giant axon to screen for dyes with large optical responses to membrane potential changes (Cohen et  al. 1974). This resulted in the identification of the merocyanine class of dyes as effective candidates for further refinement via alteration of both the chromophore and side chains (Gupta et al. 1981; Ross et al. 1977). This laboratory joined the effort to develop potentiometric dyes by applying rational design methods based on molecular orbital calculations of the dye chromophores and characterization of their binding and orientations in membranes (Loew et al. 1978, 1979a). Several important general-purpose dyes have emerged from this effort including di-5-ASP (Loew et  al. 1979b), di-4-ANEPPS (Fluhler et  al. 1985; Loew et  al. 1992), di-8-ANEPPS (Bedlack et al. 1992; Loew 1994), and di-4-ANEPPDHQ (Fisher et al. 2008; Obaid et al. 2004). All of these dyes provide rapid absorbance and fluorescence responses to membrane potential and are therefore capable of recording action potentials. They have chromophores that are in the general structural category called hemicyanine or styryl dyes. The characteristics of this class of chromophores will be discussed in detail in the next section. In addition, we have developed TMRM and TMRE (Ehrenberg et al. 1988), which are slow responding dyes that are capable of measuring smaller changes in plasma membrane potential or mitochondrial potential via confocal imaging (Farkas et  al. 1989; Loew 1993). But the primary focus of this chapter, in keeping with the theme of the book, is on fast dyes that are designed to image electrical activity in excitable cells. All of the above-mentioned dyes and several others from this lab are now available in the Molecular Probes/Invitrogen catalog, and some are also distributed by smaller companies. The dyes have been of great utility to neuroscientists interested in mapping patterns of electrical activity in complex neuronal ­preparations with numerous examples spanning the past 20 years (Djurisic et  al. 2003; Grinvald and Hildesheim 2004; Wu et  al. 1998). Interestingly, our styryl dyes have been of increasing utility

when microinjected in single cells (Antic et al. 2000; Antic 2003; Canepari et al. 2007, 2008; Djurisic and Zecevic 2005; Kampa and Stuart 2006; Milojkovic et al. 2005; Nishiyama et al. 2008; Palmer and Stuart 2009; Stuart and Palmer 2006; Zecevic 1996; Zhou et al. 2007, 2008) to follow patterns of electrical activity along very thin dendrites where direct electrical recording is precluded. Interestingly, the dyes developed in our laboratory, primarily di-4ANEPPS, have also become the standard for optical mapping of electrical activity in studies of cardiac activity (Efimov et al. 2004; Loew 2001). More recently, we have synthesized voltage-sensitive dyes (VSDs) with new hemicyanine chromophores that have absorbance and emission further toward the red end of the visible spectrum (Kee et al. 2008; Matiukas et al. 2006, 2007; Wuskell et al. 2006; Yan et  al. 2008; Zhou et  al. 2007). These dyes are often called “blue dyes” because by absorbing long-wavelength red light they appear blue. The longer wavelength absorbance and emission properties of these dyes make them useful for experiments where absorbance by endogenous chromophores, notably hemoglobin, needs to be avoided. Long wavelength also permit recording from deeper within tissue because light scattering, which limits the depth of optical penetration, is dependent on inverse fourth power of the wavelength. There are several other labs that are currently actively working on synthesizing new potentiometric dyes. Rina Hildesheim has been working for about 25 years in the laboratory of Amiram Grinvald and has synthesized hundreds of dyes. The latest dyes to emerge from these efforts are RH1691 and RH1692, oxonols that absorb at around 630 nm (Derdikman et  al. 2003; Shoham et  al. 1999; Slovin et  al. 2002). They have been successfully used for in vivo studies on awake animals. The relative response (DF/F) to electrical activity in mammalian brains for these dyes is ca. 10–3, but it is hard to compare this to other dyes as no controlled voltage clamp studies have been reported on isolated cells or membranes. The dyes have become available through Optical Imaging, Inc., a company founded by Dr. Grinvald. The laboratory of Peter Fromherz has long been interested in the photophysical properties of the hemicyanine chromophores (also called “styryl” dyes) that have been developed in this lab. Recently, they have developed a new chromophore series, the “ANINEs” that are hemicyanines enclosed in a completely rigid annelated ring framework. ANINE-6 has been particularly promising (Kuhn and Fromherz 2003; Kuhn et al. 2004)

Leslie M. Loew  •  Department of Cell Biology, R. D. Berlin Center for Cell Analysis and Modeling, University of Connecticut Health Center, Farmington, CT 06030-1507, USA M. Canepari and D. Zecevic (eds.), Membrane Potential Imaging in the Nervous System: Methods and Applications, DOI 10.1007/978-1-4419-6558-5_2, © Springer Science+Business Media, LLC 2010

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Chapter 2    L.M. Loew ●

and a new version, ANINE-6P, has improved solubility so that it may be more readily used in neuroscience applications (Fromherz et al. 2008). They are both excited at shorter wavelength than di-4-ANEPPS. Dr. Fromherz was kind enough to provide us with a sample of both dyes; in some combined Second Harmonic Generation (SHG) and 2-photon-excited fluorescence (2PF) experiments on voltageclamped cells, they show sensitivities similar to d-4-ANEPPS, but lower than the high responses we get from our new long-wavelength chromophores. However, a full characterization on our hemispherical bilayer apparatus was not possible with the quantities provided. It should also be mentioned that a recent paper (Salama et al. 2005) representing a collaboration between Alan Waggoner and Guy Salama in Pittsburgh reports on some cardiac experiments with probes that use the same longer wavelength chromophores that we have developed over the last several years. An exciting new approach incorporates green fluorescent protein into engineered channel proteins (Ataka and Pieribone 2002; Guerrero et  al. 2002; Sakai et  al. 2001; Siegel and Isacoff 1997). This approach shows great promise because of the specificity with which the constructs can be targeted to specific cells or subcellular regions. However, these probes have been either too slow or too insensitive to be practical alternatives to the organic potentiometric dyes and they are generally not properly inserted into the plasma membrane (Baker et al. 2007). Most recently, however, a significant improvement has been reported for a construct that does insert into the plasma membrane of cultured cells (Dimitrov et al. 2007) and produces reasonable sensitivities of 2%/100 mV and a time constant for response to potential steps of 20 ms. More recently, a promising fluorescence resonance energy transfer (FRET)-based dual fluorescent protein sensor has been developed with a reported 40% sensitivity/100 mV (Tsutsui et al. 2008). Whether these proteins can be targeted to specific cells in transgenic mice and whether they can continue to be improved to become practical tools for mapping potential remains to be seen. This technology will not, however, replace dyes for either the single cell microinjection experiments or for global mapping of all neurons in a macro view experiment. It is also worth pointing out that although fluorescent proteins for calcium sensing have been available for many years and continue to be improved, they are still too insensitive to have replaced dyes in the overwhelming majority of neuroscience and cardiac experiments. Our plans to improve the photostability and delivery of the potentiometric dyes should enable an even greater assortment of experiments for mapping electrical activity that will surely complement the studies that may be enabled by fluorescent protein voltage sensors.

2.2  Mechanisms of Dye Optical Responses to Changes in Membrane Potential It is important to appreciate how VSDs work as voltage sensors in order to apply them intelligently to different experimental situations. The dyes all respond by some change in their spectral properties in response to a change in membrane potential, so a prerequisite to understanding the possible mechanisms by which they can respond, is an understanding of the physical chemistry underlying the absorption and emission of a photon. The core of any dye is the chromophore – the portion of the molecule that actually interacts with the light. The absorption of a photon is possible when the difference in energy between the ground state of the chromophore and an excited state matches the energy of the photon, which is given by h/l, Planck’s constant divided by the wavelength of the light. The range of wavelengths

that can excite the molecule is broad because of the existence of vibrational sublevels within the chromophore’s electronic states and, importantly, a range of possible interactions of the chromophore with its molecular environment. Once the molecule is promoted to an excited electronic state, it immediately relaxes to the lowest energy through vibrational relaxation processes. But it stays in this vibrationally relaxed, electronically excited, state for some time, usually a few nanoseconds, before losing its energy either by emitting a photon (fluorescence) or through heat to regenerate the ground state. The fact that the excited state has had a chance to relax before it emits a photon, possibly adopting a ­conformation that is most stable for the state’s distribution of electrons and reorganizing the surrounding molecules to the most stable configuration, invariably produces a longer wavelength (lower energy) fluorescence emission spectrum compared to the band of wavelengths that is used to excite a given chromophore. It is also possible for the excited state to undergo a chemical change which would destroy the chromophore; this is generally known as bleaching. It is possible to engineer dyes so that an electric field can either interact with the chromophore’s electron distribution directly or cause the dye to change its environment. For such dyes, a change in membrane voltage can alter the absorbance or fluorescence spectrum. Good VSDs have chromophores that are bright (efficient excitation and emission), are highly sensitive to environment, and are photostable (i.e., resistant to photobleaching). There are three established mechanisms by which dyes can respond to action potentials because of a change in their molecular environment: ON–OFF, reorientation, and FRET. The ON–OFF, reorientation, and FRET mechanisms all involve a change in the location of a charged dye as a result of the changing membrane voltage (Fig. 2.1). For the ON–OFF mechanism, the dye moves from the aqueous extracellular medium to the cell membrane; because of their environmental sensitivity, the dyes will typically display a substantial increase in fluorescence upon association with the membrane and this is how the voltage change becomes transduced to a fluorescence change. Dyes with cyanine and oxonol chromophores often utilize this mechanism (Waggoner et al. 1977). A problem with this mechanism is that although the sensitivity is large, the response time of the system is often too slow to record action potentials. In the reorientation mechanism, the changing electric field within the membrane causes a membrane-bound dye to flip from an orientation perpendicular to the cell surface to an orientation where its long axis is parallel to the surface. Again therefore, the molecular environment of the dye is changed and this produces a change in the spectral properties; moreover, there is also a change in the average orientation of the dye molecules with respect to the propagation direction of the exciting light and this can also produce a change in the efficiency of light absorption. Merocyanine dyes have been shown to often utilize this mechanism (Dragsten and Webb 1978). This mechanism can be very fast, but the sensitivity can be low and quite variable from preparation to preparation. In the FRET mechanism, a donor fluorophore is anchored to the outer surface of the cell membrane and transfers its energy to nearby acceptor chromophore which then emits fluorescence at longer wavelength. If the acceptor is a negatively charged membrane-permeant dye, it will redistribute to the inner surface of the bilayer when the membrane depolarizes, thus reducing FRET and reducing the long-wavelength emission. This idea was demonstrated for a coumarin-congugated lipid donor and a permeant oxonol acceptor (Cacciatore et al. 1999; Gonzalez and Tsien 1997). The sensitivity can be high for this mechanism, but the requirement for the application of two dyes at relatively high concentration has impeded its widespread adoption. In all three of the cases

Design and Use of Organic Voltage Sensitive Dyes    Chapter 2 ●

FIGURE 2.1.  Mechanisms used by VSDs that can change their location in response to membrane depolarization.

illustrated in Fig. 2.1, the voltage change has to tip the equilibrium balance between two states of the dye molecule resulting in the movement of the dye to a new environment. Since the intrinsic properties of membranes are themselves quite variable and can therefore affect the equilibrium and/or the kinetics of dye relocation, a dye that is sensitive to potential in one preparation or set of experimental conditions may be completely ineffective in another. It would be preferable to have a mechanism which produces dyes with sensitivities that are more robust and reliable from one experimental situation to another. In pursuit of this goal, we have concentrated on producing dyes with chromophores that interact directly with the membrane electric field by an electrochromic mechanism (often referred to as a molecular Stark effect). The idea is explained in Fig. 2.2, which uses the results of molecular orbital calculations and qualitative resonance structures to demonstrate how a dye which has an electron rich p-system on one end and an electron deficient p-system on the other can switch its electron distribution upon absorption of a photon to produce the excited state. di-4-ANEPPS, the most popular of the VSDs to emerge from our work, provides a specific example. The chromophore of this dye changes its electron configuration upon excitation such that the charge shifts from the pyridinium nitrogen in the ground state to the amino nitrogen in the excited state. This behavior is predicted from molecular orbital calculations (Loew et al. 1978) such as the results shown at the top of Fig. 2.2. A polar group is appended to

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FIGURE 2.2.  Electrochromic mechanism of voltage sensitivity. The top shows how the electrons, and therefore the charge distribution, shifts upon excitation of a typical electrochromic dye. These images were generated from molecular orbital calculations where low electron density (i.e , regions of positive charge) are represented by bluer shades and high electron density (i.e., negative charge) is represented by redder colors. The lower portion of the figure shows resonance structures for the ground and excited states of one of the most widely used VSDs, di-4-ANEPPS. In this chromophore, the donor moiety is an aminonaphthyl group, the linker is a simple double bond and the acceptor is a pyridinium moiety.

one side of the chromophore in the form of a propylsulfonate moiety and two hydrocarbon chains are included at the opposite end. This, together with the intrinsic amphiphilicity of the chromophore, serves to anchor it in the membrane bilayer in an orientation that is approximately perpendicular to the surface. This orients the direction of the excitation-induced charge motion parallel to the electric field vector within the membrane, as depicted in the upper diagram of Fig. 2.3, where the dye is shown as if having been inserted from the outside of the cell. As a consequence, the ground and excited states are differentially stabilized by the intramembrane electric field, causing a shift in the spectrum when the membrane potential changes (bottom of Fig. 2.3). An equivalent way of thinking about this is to realize that the excitation-induced charge displacement moves along the direction of the electric field when the membrane is polarized and against the direction of the electric field when the membrane is depolarized. Experiments from voltage-clamped bilayer membranes (Fluhler et al. 1985; Loew et al. 1979a; Loew and Simpson 1981) have provided evidence that di-4-ANEPPS and related hemicyanine dyes do respond to membrane potential via an

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Chapter 2    L.M. Loew ●

FIGURE 2.3.  The shift in the spectrum of an electrochromic dye upon depolarization. The upper panel shows how the electric field in the membrane, E, can perturb the spectrum of an elecrochromic chromophore, represented by the stippled rectangles embedded in the outer surface of a membrane. The spectra shown in the lower half of this figure can represent absorbance, excitation, or emission spectra. The relative change in fluorescence, DF/F, is shown as a ­percentage change for different choices of detection wavelengths.

electrochromic mechanism; indeed, these bilayer experiments can even be used to estimate the magnitude of the excitation-induced charge redistribution (Loew and Simpson 1981). Although electrochromic dyes such as di-4-ANEPPS can produce unpredictable complications (discussed further in Sect. 2.4) we and others have been able to successfully utilize them in a very wide variety of experimental preparations (Loew et  al. 1992). Dyes designed to utilize an electrochromic mechanism come closest to being universally applicable and calibratable VSDs.

2.3  Measuring Voltage Changes with Voltage-Sensitive Dyes A number of modalities are available for measuring voltage-dependent optical changes from excitable cells and tissues. The most common is fluorescence. This is advantageous because of the high contrast and low background intrinsic to this technique and the wide availability of fluorescence and confocal microscopes. Other chapters in this book will describe the considerations required for optimizing the speed and sensitivity of the instrumentation. In this section, we will describe how the properties of the VSDs should be considered, focusing on fluorescence, but then briefly discussing absorbance and nonlinear optical imaging of membrane potential.

The voltage-dependent shift in the excitation or emission s­ pectrum to be expected from electrochromic dye is depicted in Fig. 2.3, where the dye is assumed to be bound to the extracellular surface of the membrane and undergo charge redistribution in its excited state in the direction shown in Fig. 2.2. Under these circumstances, the spectra shift to shorter wavelength upon membrane depolarization. The magnitude of the spectral shift will be linearly related to the change in potential. In most experiments using VSDs, a fixed band of wavelengths is continuously monitored, rather than attempting to scan the entire spectrum. Figure 2.3 shows that the choice of wavelength can have a drastic effect on the observed sensitivity of the dye response. Contrary to what might be an experimentalist’s first impulse, the wavelengths of maximal absorbance or emission are actually the worst choices for achieving optimal sensitivity. The best sensitivity is achieved at the wings of the ­spectra and will show changes in opposite directions at the high and low wavelength wings. Furthermore, measuring the change in the fluorescence relative to total fluorescence signal, DF/F, biases the optimal wavelengths even further to the extreme edges of the excitation and emission spectra because F, the denominator, decreases steadily at the edges. An approximately linear relationship between the voltage change and the fluorescence or absorbance change is obtained at the wings because the local slope of the spectrum is approximately constant, at least until it starts flattening out toward the very high and low ends of the spectrum. Another important consideration in choosing wavelengths, which mitigates against going too far out to the edges of the spectrum, is the signal-to-noise ratio (S/N). Essentially, the detection system must collect a sufficient number of photons per measurement so as to be able to determine the small modulation by the voltage with statistical reliability. For fluorescence measurements, the number of photons can be boosted by simply utilizing an intense excitation source; it should then be possible to pick wavelengths at the edge of the absorbance spectrum, where DF/F is maximal, without sacrificing signal. This is because the low absorbance at the spectral edge can be compensated by a higher intensity light source. This will permit a sufficient number of excitation events to occur in a sufficiently short time to be able to record action potentials. But a key consideration that prohibits applying this argument with respect to the edge of the emission spectrum is the limitation imposed by photobleaching. By detecting only a narrow band at the emission spectrum edge, most of the emitted photons will be lost, effectively wasting most of the excitation events. It would then be more probable that the dye will bleach before collecting sufficient photons for a good S/N. Therefore, the best compromise between DF/F and S/N is to use an intense narrow band excitation source at the edge of the VSD absorbance spectrum, while collecting the emitted light through a filter that will pass all wavelengths longer than the emission spectrum maximum. We and others have also taken advantage of the opposite changes in the voltage-dependent optical signals at the low and high wavelength wings of the spectra (Fig. 2.3) to implement a dual wavelength ratio detection scheme for measuring membrane potential with VSDs (Beach et al. 1996; Bullen et al. 1997; Knisley et al. 2000; Montana et al. 1989). The idea is to collect light from both wings of the spectrum and calculate the ratio of their fluorescence intensities as a measure of the voltage change. The primary advantage is that the sensitivity of the ratio is approximately equal to the additive absolute sensitivities at each wavelength. A second advantage is that the dual wavelength ratio can, in principle, be used to report the absolute value of the intramembrane electric field. A single wavelength fluorescence measurement can only report a relative change in potential because the intensity depends

Design and Use of Organic Voltage Sensitive Dyes    Chapter 2 ●

on the level of staining, which can be quite variable even along the membrane of a single neuron. The ratio effectively normalizes away any differential staining levels because the fluorescence intensity at both wavelengths will be proportional to dye density but oppositely responsive to intramembrane electric field. We introduced the idea of dual excitation wavelength imaging (Montana et al. 1989) and showed that it could be used to map the membrane potential along a neuron induced by an external electric field (Bedlack et al. 1992). Importantly, because the dye reports on the local electric field, any variations in dipole potential or surface potential may also produce variations in the ratio (Bedlack et al. 1994; Gross et al. 1994; Xu and Loew 2003); therefore, any spatial variations in ratio must be interpreted with caution and may not reflect a variation in transmembrane potential. On the other hand, fast temporal variations in the ratio are likely to reflect changes in transmembrane potential because the changes in lipid composition that would be required for changes in other sources of intramembrane electric field would be slow on the timescale of action potentials. A dual emission wavelength measurement is preferred for rapid spatial mapping of electrical activity because two fixed detection paths can be employed (Bullen et al. 1997; Bullen and Saggau 1999) rather than mechanically switching between two excitation wavelength filters. In general, dual wavelength ratiometric measurements require more complex instrumentation and analysis than simply monitoring DF/F, so the adoption of this approach has been limited to experiments that require extracting a voltage-dependent signal from other confounding variables. A commonly used alternative to fluorescence for optically monitoring electrical activity is to record or image the light transmitted through a specimen stained with a VSD (Glover et al. 2008; Sasaki et al. 2002). A change in the transmittance of a stained preparation simply reflects the change in the absorbance spectrum of the dye. Of course, the transmitted light corresponds to the light that actually does not get absorbed by the dye. The amount of light that is absorbed by a single dye-stained membrane depends on the size of the cell, the surface density of the dye molecules on the membrane, and the extinction coefficient of the dye at the chosen wavelength. But it can be estimated that this will never exceed 1 part in 103 for even the largest cell. The modulation of the transmitted light signal by an action potential would therefore never be much greater than ~1 part in 104. A comparable estimate for the modulation of fluorescence is 1 part in 10, because the entire light signal emanates from the stained membrane. So if DT/T is intrinsically so much lower than DF/F, why would anyone prefer to use transmitted light for optical electrophysiological recording? Indeed, the technique is almost never used for recording from single cells. In experiments where the collective activity of a large population of cells in a tissue needs to be imaged, the sensitivity of the technique is increased because more of the incident light is absorbed. Furthermore, the S/N in a transmitted light detection is not limited by photon statistics so simpler and less noisy photodetectors can be used. Another approach that is just beginning to emerge for VSDs is 2PF (Fisher et al. 2008; Kuhn et al. 2008; Loew et al. 2002). There is really no difference in principle in the way that 1- and 2-PF measures the dye response, except, of course, that the 2-photon modality requires a femtosecond pulsed laser operating at twice the wavelength and a microscope configuration that is optimized for the purpose (Denk et al. 1990). The major advantage of 2PF is the ability to probe deep inside a specimen with high 3D resolution. The disadvantage, in addition to complex instrumentation, is that the laser scanning required to obtain a full image of the specimen is too slow for recording of action potential activity; therefore, line scans or special spatial sampling protocols are used to record

17

optical signals from a small number of sites. Chapter11 in this book is devoted to two-photon microscopy of VSDs. Second harmonic generation is another nonlinear optical process (Campagnola and Loew 2003; Millard et  al. 2003a) that can take place in a microscope coupled to an ultrafast laser. As in the case of two-photon excitation the probability of SHG is proportional to the square of the incident light intensity. While 2PF involves the nearsimultaneous absorption of two photons to excite a fluorophore, followed by relaxation and noncoherent emission, SHG is a nearly instantaneous process in which two photons are converted into a single photon of twice the energy, emitted coherently. Furthermore, SHG is confined to loci lacking a center of symmetry; this ­constraint is readily satisfied at cellular membranes in which SHG-active constituents are unevenly distributed between the two leaflets of the lipid bilayer. The first examination of the nonlinear optical properties of VSDs was in a collaboration between this lab and Aaron Lewis’ lab, where we analyzed SHG from a monolayer of di-4-ANEPPS in a Langmuir–Blodgett trough (Huang et al. 1988). Subsequently (BenOren et al. 1996; Bouevitch et al. 1993; Campagnola et al. 1999), we were able to show that the SHG signal from electrochromic dye stained membranes is sensitive to membrane potential. These experiments were continued (Clark et al. 2000; Millard et al. 2003b, 2004, 2005a, b; Teisseyre et  al. 2007) in an attempt to characterize and optimize the SHG response of our dyes to membrane potential. They indicated that the mechanism could not be explained as a direct electrooptic effect because the kinetics of the response for some of the dyes was slow, although these were not always the conclusions reached by others (Jiang et al. 2007; Pons et al. 2003). Whatever the mechanism, the laboratories of Rafael Yuste, Kenneth Eisenthal, and Watt Webb (Araya et al. 2006, 2007; Dombeck et al. 2004, 2005; Nuriya et  al. 2006) were able to demonstrate the measurement of action potentials using second harmonic imaging microscopy. These studies used the hemicyanine dye FM-464 applied to the interior of the cell through a patch pipette. Because the SHG can only come from dye that is noncentrosymmetrically distributed on the spatial scale of the wavelength, the light emanates only from the internally stained plasma membrane and no signal appears from internally stained organelles with highly convoluted membranes such as the endoplasmic reticulum and mitochondria. Because of this elimination of background from these internal electrically inactive membranes, the voltage sensitivity of the SHG signal can approach 20%/action potential – much higher than is practically possible with fluorescence. However, these measurements had very low S/N and required extensive signal averaging because of the low intensity of the SHG. Therefore, it is not clear how generally useful this modality will ultimately be until new dyes are developed with much stronger SHG efficiencies. Chapter 13 provides a thorough review of membrane potential measurements with SHG.

2.4  Choosing the Best Dye for an Experiment Many of the hemicyanine dyes synthesized in our and other labs are capable of sensing membrane potential changes with high sensitivity. But different experimental designs require different spectral properties, solubility properties, and membrane binding affinities. Furthermore, competing processes can sometimes diminish the sensitivity of a given dye in a particular experimental preparation. So there is a need for a large repertoire of dyes to meet these varying needs and this section will consider how to make these choices. We focus on dyes developed in this laboratory as exemplars, but the same principles can be applied to the dyes developed by others.

18

Chapter 2    L.M. Loew ●

A prerequisite for choosing a dye is simply being able to appreciate the structural variety of VSDs and establishing some nomenclature that permits us to refer to them in a recognizable way. As discussed in Sect. 2.2, the hemicyanine chromophores are all characterized by one end that is electron rich in the ground state, also called an electron donor moiety, and another end that is electron deficient, an electron acceptor moiety (Fig. 2.2). When these groups are linked with a p-electron linker, it is possible for the electrons to readily shift in response to excitation. Chromophores with such electronic structures are commonly called “push-pull” chromophores because of the complementary tendencies of the electrons at the two ends. Figure 2.4 lists all of the donor, linker, and acceptor moieties that we have employed in our lab to develop push-pull chromophores. We have synthesized approximately 300 dyes utilizing over 50 of these push-pull hemicyanine chromophores. The R1 groups on the donor ends are usually hydrocarbon chains that anchor the dye to the hydrophobic interior of the membrane. The R2 groups on the acceptor ends are usually hydrophilic groups that protrude into the aqueous medium adjacent to the membrane and help to maintain the orientation of the chromophore perpendicular to the membrane surface (cf. Fig. 2.3). The laboratories of Amiram Grinvald and Alan Waggoner have also developed large numbers of VSDs using some of these groups. Table 2.1 ­provides the spectral characteristics of most of the chromophores constructed from these parts in ethanol, water, and when bound to lipid vesicle membranes. An important attribute of almost all of these dyes is that they have strong fluorescence quantum efficiencies in the membrane bound forms, but typically at least two order of magnitude lower fluorescence in aqueous solution. This means that the background signal from unbound dye can usually be completely neglected even if the preparation is allowed to remain exposed to the aqueous staining solution. For the reasons discussed in Sect. 2.3, the optimal wavelengths for voltage sensitivity are typically ~50 nm above the maximum wavelengths reported for the lipid spectra in Table 2.1. The systematic chemical names for these compounds are extremely cumbersome because of their complexity. Therefore, the field has adopted different styles of abbreviation. Most commonly, a dye is identified by the initials of the chemist who synthesized it followed by a number. For example JPW-3080 was synthesized by Joseph P. Wuskell and its synthesis was first recorded on page 080 of his third notebook. Similarly, RH-160 is dye number 160 prepared by Rena Hildesheim in Amiram Grinvald’s lab. Of course these abbreviations do not convey much information about the properties of the dyes and we have attempted to formulate more descriptive abbreviations for the more common hemicyanine VSDs. The general formulation we have adopted uses the following scheme: di-n-DLAH This provides a designation for their structural components as follows: alkyl chain lengths (n) – p-Donor – Linker(s) – p-Acceptor – hydrophilic Head group (Wuskell et al. 2006). A pair of alkyl chains, R1, is appended to the amino terminus of most of dyes (right side of each structure in Fig. 2.4). The number of carbons in these chains is indicated by n, in the “Di-n” portion of the naming scheme. The p-donor moiety, D, can be aminophenyl (AP, -1- in Fig. 2.4), aminonaphthyl (AN, 2- in Fig. 2.4), etc. The linker, L, is ethene (E, -1- in Fig. 2.4) diene (D, -2-), ethene-furan-ethene (EFE, -7-), etc. The acceptors, A, can be pyridinium (P, -1), quinolinium (Q, -3) indolenium (In, -5), etc. Finally, we have dyes with various head groups, H, the most common of which are shown in Fig. 2.5. Thus, di-4ANEPPS in Fig. 2.2 has a pair of butyl groups (n = 4) attached to the amino group of an aminonaphthyl donor (D = AN), which is linked

FIGURE 2.4.  The components that have been used to construct hemicyanine push-pull chromophores. A combinatorially large repertoire of chromophores have been constructed by mixing the D–L–A moieties. The abbreviations for some of the more common components are shown alongside the number scheme that was employed to designate the chromophores in Table 2.1. VSDs are then realized from the chromophores by appending hydrophobic tails, R1, and hydrophilic head groups, R2.

via an ethene linker (L = E) to a pyridinium (A = P) acceptor, which is appended with a propylsulfonate (H = PS) head group. The chromophore determines the optical properties of the VSD (Table 2.1) and also its intrinsic voltage sensitivity. We screen every new dye using a voltage-clamped hemispherical bilayer apparatus (Fluhler et al. 1985; Loew and Simpson 1981; Wuskell et al. 2006). From these experiments, as well as experience in other preparations

Design and Use of Organic Voltage Sensitive Dyes    Chapter 2 ●

19

TABLE 2.1.  Wavelength Ranges of Absorbance and Emission Maxima for Styryl Dye Chromophores Chromophore [1–1–1] [1–1–2] [1–1–3] [1–1–4] [1–1–5] [1–1–6] [1–2–1] [1–2–3] [1–2–4] [1–2–5] [1–3–1] [1–4–1] [1–5–4] [1–6–1] [1–7–3] [1–8–3] [1–8–5] [1–9–3] [1–9–5] [1–10–3] [1–11–1] [1–11–4] [2–1–1] [2–1–7] [2–2–3] [2–2–6] [3–1–3] [3–1–4] [4–1–3] [4–1–4] [4–1–6] [4–7–3] [5–1–3] [6–1–1] [6–1–3] [6–1–5] [7–1–1] [7–1–3] [7–1–4] [7–1–5] [7–1–6] [7–2–4] [7–8–3] [7–9–3] [8–1–1] [8–1–3] [9–1–1] [9–1–2] [9–1–3]

Number

lABS (EtOH)

lEM (EtOH)

lABS (lip)

lEM (lip)

18 2 3 3 1 2 5 2 3 1 1 1 2 1 3 1 1 4 2 1 1 1 47 1 1 1 3 1 1 3 1 1 1 1 2 1 1 3 4 1 1 1 1 2 1 1 1 2 2

490 ± 9 470 564 ± 5 564 ± 5 567 590 ± 1 510 ± 10 560 ± 30 700 ± 20 650 475 485 630 ± 10 482 640 ± 20 607 626 590 ± 20 690 ± 30 626 440 391 500 ± 10 486 584 609 580 ± 6 681 608 705 ± 7 610 592 514 474 520 ± 20 616 523 620 ± 10 726 ± 10 588 600 825 663 620 ± 30 440 500 472 540 ± 20 540 ± 20

617 ± 8 610 ± 10 684 ± 7 684 ± 7 599 679 680 ± 50 675 ± 7 800 ± 50 700 610 715 720 ± 40

468 ± 7 441 ± 2 533 ± 5 533 ± 5 562 578 468 ± 7 540 ± 20 611 ± 10 638 450 463 580 ± 10 461 580 ± 10 579 634 548 ± 5 640 ± 10 616 420 404 470 ± 10 464 526

600 ± 10 590 ± 30 680 ± 70 680 ± 70 601 662 620 ± 20

including squid axon and lobster nerve (Loew et al. 1992; Wuskell et al. 2006), the chromophores with the best intrinsic ­voltage sensitivity are (DLA): APEP, ANEP, ANEQ, ANBDQ, APEThEQ, ANEThEQ, ANBDIn, APEThEIn, and ANEThEIn. These chromophores span a range of over 200 nm in optimal excitation and emission wavelengths. In particular, the more recently developed long-wavelength VSDs have become increasingly important in applications involving multiple optical probes, deeper tissue penetration, or avoidance of interference from intrinsic ­chromophores such as blood (Kee et al. 2008; Matiukas et al. 2006, 2007; Zhou et al. 2007, 2008). For example, in a collaboration with the lab of George Augustine, we have shown that di-2-ANBDQPQ is a

890 ± 70 639 800 ± 100 870 664 641 692 710 ± 10 812 689 ± 1 770 833 833 873 620 656 714 ± 9 636 640 720 ± 10 810 ± 40 612 708 880 687 588 648 672 760 ± 40 760 ± 40

771 ± 1 584 690 660 ± 10 670 740 ± 30 646 647 720 ± 50 757 650 552 640 ± 20 627 702

556 ± 5 658 589 612 ± 1 610 597 500 432 480 ± 20 620

680

579 ± 1 678 ± 7 594 618 674 668 559 421 486 457 530 ± 30 530 ± 30

673 ± 4 770 ± 60 618

760 716 715 714 630 671 ± 8 643

790 680 566 640 622 681 ± 8 681 ± 8

good long-wavelength dye for recording from hippocampal slices. Figure 2.6 shows the results of one of these voltage imaging experiments (Kee et  al. 2008). The excitation spectra of these ­long-wavelength dyes also avoid those of light-activated proteins used to control neuronal activity, such as channelrhodopsin-2 and halorhodopsin (Zhang et al. 2007), making it possible to combine the use of VSDs with such proteins. But whether a given VSD is successful in a specific experimental application also depends critically on the side chains and head groups. The choice of alkyl chains provides direct control of the solubility and membrane binding characteristics. Short alkyl chains impart good water solubility at the expense of strong membrane binding;

20

Chapter 2    L.M. Loew ●

FIGURE 2.5.  Structures and abbreviations of commonly utilized polar head groups corresponding to R2 in Fig. 2.4.

this would be appropriate for staining a thick tissue where the dye needs to penetrate many cell layers. However, VSDs with greater solubility can also be more readily washed out of a preparation. We have found that longer R1 alkyl chains increase the strength of binding of the dyes to the membrane and also slow the rate of internalization. Internalization is detrimental to the potentiometric response because as the dye equalizes on either side of the bilayer the voltage-dependent spectral shifts from the two leaflets will cancel each other (cf. Fig. 2.2). However, long side chains decrease the solubility of the dye, making staining slow and ­inefficient and impractical for thick tissue. This problem can be ameliorated through the use of vehicles such as Pluronic F127 (Lojewska and Loew 1987) or g-cyclodextrin (Wuskell et  al. 2006), which form complexes with the dyes to keep them d­ isbursed in solution. With respect to R2, the negatively charged sulfonate head groups (PS and BS in Fig. 2.5) are attractive because they provide internal counter ions for the positive charge of the chromophore, resulting in an overall neutral VSD. These are less likely to interact and possibly interfere with other cellular components. The positively charged head groups in Fig. 2.5 provide some additional water solubility and can therefore partially offset the solubility lowering effect of moderately sized R1 side chains. Indeed, di-3-ANEPPDHQ and di-4-ANEPPDHQ have proven to be excellent general purpose dyes for staining of multicellular preparations (Obaid et al. 2004). Similarly, several of the new long-wavelength dyes with positive head groups can be used for labeling for recording of large neuronal populations in brain slices (di-2-ANBDQPQ in Fig. 2.6) (Kee et al. 2008). An important finding for the doubly positively charged VSDs with the positive head groups is that, given enough time, they can become internalized and will be retained in the cell interior because of the polarized resting potential. This has led to applications where long R1 side chain dyes with the PQ head group are applied in an ethanol solution to cause the dyes to precipitate out and remain localized in a confined anatomical region. They then can become internalized and be transported over a period of hours by retrograde flow or diffusion to regions of the neurons remote from where they were originally stained. This strategy was first employed with di-8-ANEPPQ and di-12-ANEPEQ where the dye was applied to chick spinal cord to ultimately label and record from remote neuronal cell bodies, dendrites, and axons (Tsau et al. 1996; Wenner et al. 1996). When the positively charged head groups are combined with R1 chains of just one or two carbons in length, the resulting VSDs are extremely water soluble, often at a solubility of several mg/ml. This is important for applications where the dyes must be applied internally to a single cell through a patch pipette. Such experiments are

FIGURE 2.6.  Spatio-temporal features of responses reported by di-2-ANBDQPQ following simulation of a mouse hippocampal brain slice. The fluorescence was excited with 615–685 nm light and emission was collected at >700 nm. (A) Pseudocolor map of the response measured at its peak (8.8 ms after stimulus). Numbers refer to positions where the responses shown in (B) were recorded, both across the SR (positions 1–5) and transecting the CA1 layers (positions 3 and 6–9). (B) Optical signals recorded at multiple locations; each response was measured from a single pixel at the numbered sites [reprinted from Kee et al. (2008) with kind permission from Springer Science+Businness Media].

designed to allow imaging of membrane potential from different regions of a single neuron in a brain slice or in vivo (Antic et al. 1999; Antic and Zecevic 1995; Antic 2003; Canepari et al. 2007; Milojkovic et  al. 2005; Palmer and Stuart 2009; Stuart and Palmer 2006; Zhou et al. 2007, 2008). For example, Palmer and Stuart (2009) have been able to image membrane potential from

Design and Use of Organic Voltage Sensitive Dyes    Chapter 2 ●

single dendritic spines with di-1-ANEPEQ and Zhou et al. (2008) have been able to image back propagating action potentials with di-2-ANBDQPQ.

2.5  How Can Better Dyes Expand The Scope of Possible Experiments? One major area for improvement is dye photostability. Obviously, more photostable dyes will permit longer duration recording ­experiments; but as importantly, they will permit high excitation intensities with a resultant higher emitted photon flux. This is important because the S/N for recording fast electrical signals is limited by the stochastic shot noise of the number of photons that can be collected – the S/N is proportional to (photon count)1/2. Higher dye brightness (the product of extinction coefficient and fluorescence quantum efficiency) will also help in the same way. Greater sensitivity of the optical signal to voltage changes would expand the range of applications for VSDs. We have produced dyes that can give tenfold or more fluorescence changes for a 60 mV change in potential (Ehrenberg et al. 1988; Loew et al. 1983), but these dyes depend on a voltage-dependent redistribution across the membrane that renders the response too slow to follow an action potential. Fast dyes necessarily have much smaller voltage sensitivities. The best of our fast dyes, when excited at the spectral edge, can produce relative fluorescence changes of ~40%/100 mV. Frankly, we do not anticipate much improvement in this sensitivity, although improved instrumentation can further stretch the range of experiments accessible to the technology. On the other hand, SHG represents a relatively unexplored optical modality that could produce much greater sensitivity than fluorescence and this is the rationale for our continued interest in developing this technique despite the S/N limitation. Our reasoning is that by resolving the mystery of how SHG responds to potential, we should be able to use this understanding to significantly improve the already high sensitivity by optimizing the design of the dyes and the optics. Dyes, light sources, and optical modalities are required to ­permit deeper penetration into thick tissue or in  vivo imaging. Longer wavelength dyes will allow for deeper penetration because their spectra are removed from interference by endogenous chromophores and because light scattering diminishes as the inverse of the fourth power of wavelength. Nonlinear optical modalities like 2PF and SHG will allow deeper optical sectioning if they can be implemented with longer wavelength ultrafast pulsed lasers. One major area for future chemistry is to devise methods for more specific delivery of the dyes to individual, preferably multiple, cells in a complex preparation. Indeed, this is one of the main motivations for the intense activity in trying to devise a genetically encodable fluorescent protein voltage sensor. But by combining engineered organic dyes, photochemistry, and new optical technologies, it should be possible for chemists to devise general solutions to this challenge as well.

Acknowledgment I am indebted to the many talented chemists, microscopists, and neuroscientists who have collaborated with me and who have carried out much of the research summarized in this chapter. Most notably, I wish to acknowledge my long-term collaborators Larry Cohen, Aaron Lewis, Mei-de Wei, and Joe Wuskell. The more recent work in my lab has benefited from collaborations with Ping Yan and Srdjan Antic. This work was supported by NIH EB001963.

21

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Chapter 2    L.M. Loew ●

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Loew LM (1994) Voltage sensitive dyes and imaging neuronal activity. Neuroprotocols 5:72–79. Loew LM (2001) Mechanisms and principles of voltage sensitive fluorescence. In Rosenbaum DS, Jalife J (eds) Optical mapping of cardiac excitation and arrhythmias. Futura Publishing, Armonk. Loew LM, Simpson L (1981) Charge shift probes of membrane potential. A probable electrochromic mechanism for ASP probes on a hemispherical lipid bilayer. Biophys J 34:353–365. Loew LM, Bonneville GW, Surow J (1978) Charge shift optical probes of membrane potential theory. Biochemistry 17:4065–4071. Loew LM, Scully S, Simpson L, Waggoner AS (1979a) Evidence for a chargeshift electrochromic mechanism in a probe of membrane potential. Nature 281:497–499. Loew LM, Simpson L, Hassner A, Alexanian V (1979b) An unexpected blue shift caused by differential solvation of a chromophore oriented in a lipid bilayer. J Am Chem Soc 101:5439–5440. Loew LM, Rosenberg I, Bridge M, Gitler C (1983) Diffusion potential cascade. Convenient detection of transferable membrane pores. Biochemistry 22:837–844. Loew LM, Cohen LB et al (1992) A naphthyl analog of the aminostyryl pyridinium class of potentiometric membrane dyes shows consistent sensitivity in a variety of tissue, cell, and model membrane preparations. J Membr Biol 130:1–10. Loew LM, Campagnola P, Lewis A, Wuskell JP (2002) Confocal and nonlinear optical imaging of potentiometric dyes. Methods Cell Biol 70:429–452. Lojewska Z, Loew LM (1987) Insertion of amphiphilic molecules into membranes is catalyzed by a high molecular weight non-ionic surfactant. Biochim Biophys Acta 899:104–112. Matiukas A, Mitrea BG et al (2006) New near-infrared optical probes of cardiac electrical activity. Am J Physiol Heart Circ Physiol 290: H2633–H2643. Matiukas A, Mitrea BG et al (2007) Near infrared voltage sensitive fluorescent dyes optimized for optical mapping in blood-perfused myocardium. Heart Rhythm 4:1441–1451. Millard AC, Campagnola PJ, Mohler W, Lewis A, Loew LM (2003a) Second harmonic imaging microscopy. In Marriott G, Parker I (eds) Methods in enzymology, vol 361B. Academic Press, San Diego. Millard AC, Jin L, Lewis A, Loew LM (2003b) Direct measurement of the voltage sensitivity of second-harmonic generation from a membrane dye in patch-clamped cells. Opt Lett 28:1221–1223. Millard AC, Jin L et al (2004) Sensitivity of second harmonic generation from styryl dyes to trans-membrane potential. Biophys J 86:1169–1176. Millard AC, Jin L et al (2005a) Wavelength- and time-dependence of potentiometric non-linear optical signals from styryl dyes. J Membr Biol 208:103–111. Millard AC, Lewis A, Loew LM (2005b) Second harmonic imaging of membrane potential. In imaging in neuroscience and development. In: Yuste R, Lanni F, Konnerth A (eds) Imaging neurons a laboratory manual. Cold Spring Harbour Laboratory Press, New York. Milojkovic BA, Wuskell JP, Loew LM, Antic SD (2005) Initiation of sodium spikelets in basal dendrites of neocortical pyramidal neurons. J Membr Biol 208:155–169. Montana V, Farkas DL, Loew LM (1989) Dual-wavelength ratiometric fluorescence measurements of membrane potential. Biochemistry 28: 4536–4539. Nishiyama M, von Schimmelmann MJ, Togashi K, Findley WM, Hong K (2008) Membrane potential shifts caused by diffusible guidance signals direct growth-cone turning. Nat Neurosci 11:762–771. Nuriya M, Jiang J, Nemet B, Eisenthal KB, Yuste R (2006) Imaging membrane potential in dendritic spines. Proc Natl Acad Sci U S A 103:786–790. Obaid AL, Loew LM, Wuskell JP, Salzberg BM (2004) Novel naphthylstyrylpyridinium potentiometric dyes offer advantages for neural network analysis. J Neurosci Methods 134:179–190. Palmer LM, Stuart GJ (2009) Membrane potential changes in dendritic spines during action potentials and synaptic input. J Neurosci 29: 6897–6903. Pons T, Moreaux L, Mongin O, Blanchard-Desce M, Mertz J (2003) Mechanisms of membrane potential sensing with second-harmonic generation microscopy. J Biomed Opt 8:428–431.

Design and Use of Organic Voltage Sensitive Dyes    Chapter 2 ●

Ross WN, Salzberg BM et al (1977) Changes in absorption, fluorescence, dichroism, and birefringence in stained giant axons: optical measurement of membrane potential. J Membr Biol 3:141–183. Sakai R, Repunte-Canonigo V, Raj CD, Knopfel T (2001) Design and characterization of a DNA-encoded, voltage-sensitive fluorescent protein. Eur J Neurosci 13:2314–2318. Salama G, Choi BR et al (2005) Properties of new, long-wavelength, voltagesensitive dyes in the heart. J Membr Biol 208:125–140. Sasaki S, Yazawa I et al (2002) Optical imaging of intrinsic signals induced by peripheral nerve stimulation in the in  vivo rat spinal cord. Neuroimage 17:1240–1255. Shoham D, Glaser DE et al 1999. Imaging cortical dynamics at high spatial and temporal resolution with novel blue voltage-sensitive dyes. Neuron 24:791–802. Siegel MS, Isacoff EY (1997) A genetically encoded optical probe of membrane voltage. Neuron 19:735–741. Slovin H, Arieli A, Hildesheim R, Grinvald A (2002) Long-term voltage-sensitive dye imaging reveals cortical dynamics in behaving monkeys. J Neurophysiol 88:3421–3438. Stuart GJ, Palmer LM (2006) Imaging membrane potential in dendrites and axons of single neurons. Pflugers Arch 453:403–410. Teisseyre TZ, Millard AC et al (2007) Nonlinear optical potentiometric dyes optimized for imaging with 1064-nm light. J Biomed Opt 12:044001. Tsau Y, Wenner P et al (1996) Dye screening and signal-to-noise ratio for retrogradely transported voltage-sensitive dyes. J Neurosci Methods 170: 121–129. Tsutsui H, Karasawa S, Okamura Y, Miyawaki A (2008) Improving membrane voltage measurements using FRET with new fluorescent proteins. Nat Meth 5:683–685.

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Waggoner AS, Wang CH, Tolles RL (1977) Mechanism of potential-dependent light absorption changes of lipid bilayer membranes in the presence of cyanine and oxonol dyes. J Membr Biol 33:109–140. Wenner P, Tsau Y, Cohen LB, O’Donovan MJ, Dan Y (1996) Voltage-sensitive dye recording using retrogradely transported dye in the chicken spinal cord: staining and signal characteristics. J Neurosci Methods 170:111–120. Wu JY, Lam YW et  al (1998) Voltage-sensitive dyes for monitoring multineuronal activity in the intact CNS. Histochem J 30:169–187. Wuskell JP, Boudreau D et al (2006) Synthesis, spectra, delivery and potentiometric responses of new styryl dyes with extended spectral ranges. J Neurosci Methods 151:200–215. Xu C, Loew LM (2003) The effect of asymmetric surface potentials on the intramembrane electric field measured with voltage-sensitive dyes. Biophys J 84:2768–2780. Yan P, Xie A, Wei MD, Loew LM (2008) Amino(oligo)thiophene-based ­environmentally sensitive biomembrane chromophores. J Org Chem 73: 6587–6594. Zecevic D (1996) Multiple spike-initiation zones in single neurons revealed by voltage-sensitive dyes. Nature 381:322–325. Zhang F, Aravanis AM, Adamantidis A, de Lecea L, Deisseroth K (2007) Circuit-breakers: optical technologies for probing neural signals and systems. Nat Rev Neurosci 8:577–581. Zhou W-L, Yan P, Wuskell JP, Loew LM, Antic SD (2007) Intracellular longwavelength voltage-sensitive dyes for studying the dynamics of action potentials in axons and thin dendrites. J Neurosci Methods 164:225–239. Zhou WL, Yan P, Wuskell JP, Loew LM, Antic SD (2008) Dynamics of action potential backpropagation in basal dendrites of prefrontal cortical pyramidal neurons. Eur J Neurosci 27:923–936.

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Imaging Submillisecond Membrane Potential Changes from Individual Regions of Single Axons, Dendrites and Spines Marco Canepari, Marko Popovic, Kaspar Vogt, Knut Holthoff, Arthur Konnerth, Brian M. Salzberg, Amiram Grinvald, Srdjan D. Antic, and Dejan Zecevic 3.1  Introduction Understanding the biophysical properties and functional organization of single neurons and how they process information is fundamental to understanding how the brain works. Because the primary function of any nerve cell is to process electrical signals [i.e., membrane potential (Vm) transients], usually from multiple sources, there is a need for detailed spatiotemporal analysis of electrical events in thin axonal and dendritic processes. This requirement resulted in the development of new measurement techniques that allow monitoring the electrical activity of different parts of the same cell simultaneously. A major experimental advance in this field, which also underscored the importance of such measurements, was achieved by the development of the recording method that made possible simultaneous monitoring of voltage transients from two or more dendritic locations on a single neuron (multiple patch-electrode recording in brain slices; Stuart et  al. 1993; Stuart and Sakmann 1994). More recently, methods were developed for patch-pipette recording of electrical signals from cut ends of axons of layer 5 pyramidal cells in the cerebral cortex (Shu et al. 2006) as well as from presynaptic axon terminals of the giant synapses of mossy fiber-CA3 connections (Bischofberger et al. 2006). These techniques, however, are still limited in their capacity for assessing spatiotemporal patterns of signal initiation and propagation in complex dendritic and axonal processes. Moreover, many subcellular structures including small diameter terminal dendritic branches as well as dendritic spines and most axon terminals and axon collaterals are not accessible to electrodes. To overcome these limitations, it was highly desirable to complement the patch-­electrode approach with technologies that permit extensive parallel recordings from all parts of a neuron with adequate spatial and temporal resolution. An adequate temporal resolution in recording neuronal action potential (AP) and synaptic potential signals is in the submillisecond range, as determined by the duration of different phases of the AP and synaptic potential (SP) waveforms. An adequate spatial resolution is on the order of 1 mm, as determined by the dimensions of neuronal terminal processes and dendritic spines. This spatiotemporal resolution can now be realized using optical recording of Vm changes with organic voltage-sensitive dyes

(Vm imaging). The sensitivity of this measurement technique has recently reached a level that permits single trial optical recordings of Vm transients from all parts of a neuron, including axon terminals and collaterals, terminal dendritic branches, and individual dendritic spines. Whenever the experimental design allows signal averaging, a relatively small number of trials (4–9) will result in two or threefold improvements in the signal-to-noise ratio (S/N).

3.2  Determinants Of Vm-Imaging Sensitivity The central figure of merit in Vm imaging is the recording sensitivity expressed as the S/N. The rules governing the sensitivity of light intensity measurements are well understood and may be put in a nutshell with an expression:

S / N ¥ ( DF/F ) ÖF,

(3.1)

where DF/F is the fractional fluorescence signal per unit change in Vm and F is the number of detected photons per unit time (fluorescence intensity expressed as photon flux). This relationship is valid under the shot noise limited conditions fulfilled in most modern fluorescence measurements. Shot noise limited conditions imply that the noise arising from the statistical nature of the emission and detection of photons (shot noise) is the dominant source of noise in the recording system, while the following sources of noise are ­negligible: (a) the noise in the incident light intensity arising from an unstable light source (50 or 60 Hz and harmonic ripple noise and/or arc-wander noise); (b) noise caused by mechanical vibration of the image projected on the photodetector; (c) electrical noise in the amplifier circuits (dark noise); and (d) read noise of the CCD camera (see also Chap. 1). Under these conditions, one way to increase the S/N for a given dye is to increase F by increasing either the incident light intensity or the detection efficiency, or both. An additional possibility is to increase the fractional fluorescence change (DF/F) per unit change in Vm (sensitivity of the dye). Thus, close attention must be paid to these two parameters.

Marco Canepari, Kaspar Vogt  •  Division of Pharmacology and Neurobiology, Biozentrum – University of Basel, Basel, Switerland Marko Popovic and Dejan Zecevic  •  Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, CT 06520, USA Knut Holthoff  •  Hans Berger Klinik für Neurologie, Friedrich-Schiller-Universität Jena, Jena, Germany Arthur Konnerth  •  Center for Intergrated Protein Science and Institute of Neuroscience, Technical University Munich, Munich, Germany Brain M. Salzberg  •  Departments of Neuroscience and Physiology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania Amiram Grinvald  •  Department of Neurobiology, Weizmann Institute of Science, Rehovot 76100, PO Box 26, Israel Srdjam D. Antic  •  Department of Neuroscience, UConn Health Center, Farmington, CT 06030, USA M. Canepari and D. Zecevic (eds.), Membrane Potential Imaging in the Nervous System: Methods and Applications, DOI 10.1007/978-1-4419-6558-5_3, © Springer Science+Business Media, LLC 2010

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The detected fluorescence photon flux F is a function of ­several parameters. These include: (a) the excitation light intensity; (b) the extinction coefficient of the dye absorption; (c) the quantum yield of a given dye (the efficiency of the fluorescence process defined as the ratio of the number of photons emitted to the number of ­photons absorbed); (d) the overall light throughput of the optical elements in the light path, including objectives, mirrors, auxiliary lenses, and optical filters; (e) the quantum efficiency of the recording device (the ratio between electrons generated to the number of photons absorbed at the photoreactive surface). In addition to these factors that are usually constant for a given experiment, the photon flux will also depend on three additional variables: (f) the number of membrane-bound dye molecules in the light path which will be a function of the amount of staining and the membrane surface area projected onto individual pixels of the recording device; (g) the fraction of the fluorescent dye bound to the external membrane that changes potential; and (h) the time interval over which photons are collected for each data point as determined by the imaging frame rate. Thus, the photon flux and S/N will decrease as the spatial and temporal resolutions are increased. The voltage sensitivity of the dye, expressed as the relative fluorescence change (DF/F), depends on its chemical structure as described in Chap. 2. In addition, for a given dye, the voltage sensitivity is a function of the excitation wavelength, as demonstrated early in the development of organic probes (Cohen et  al. 1974; Loew 1982). The sensitivity increases at extreme wings of the absorption spectrum for charge-shift voltage-sensitive probes. Also, the optimal sensitivity will be reached when all excitation occurs for the wavelength with the best response. In other words, monochromatic illumination at the appropriate wavelength (as opposed to the conventional use of a bandpass interference filter to select a range of wavelengths) will result in optimal sensitivity (Kuhn et al. 2004). These considerations argue that a laser emitting monochromatic light at an appropriate wavelength will be the optimal excitation light source. It should be noted that the advantages of laser illumination are more pronounced in fluorescent voltagesensitive dye measurements since the loss of coherence introduced by fluorescence emission eliminates the speckle noise that plagues transmission (absorption) measurements using laser sources (Dainty 1984; Gratton and vande Ven 1989).

3.3  Foundation of Presently Available Recording Sensitivity The feasibility of multiple site optical recording from individual nerve cells was initially demonstrated using monolayer neuronal culture and extracellular application of the voltage-sensitive dye (Grinvald et  al. 1981). It was subsequently shown that sufficient sensitivity of recording with subcellular spatial resolution from intact parts of the nervous system can only be achieved if nerve cells are labeled selectively by the intracellular application of the membrane impermeant probe. This section reviews the limits to the sensitivity of recording using extracellular and intracellular application of the voltage-sensitive dyes.

in measurements from dissociated neurons in monolayer culture. Both absorption and fluorescence measurements were used in these experiments and the individual dissociated neurons in a culture dish were stained from the extracellular side by bath application of the dye (Grinvald and Farber 1981). Typical optical recordings obtained from absorption and fluorescence measurements using the most sensitive probes available at the time are shown in Fig. 3.1. It has also been possible, using the same approach, to study synaptic interactions between several interconnected neurons in culture (Parsons et al. 1989, 1991), and the cell-to-cell propagation of the AP in patterned growth cardiac myocytes forming two-dimensional hearts in culture (Rohr and Salzberg 1994). The monolayer neuronal culture is a low opacity system especially convenient for the extracellular selective staining of the outside cellular membrane as well as for both absorption and fluorescence ­measurements. However, primary cultures are networks of neurons that grow under artificial conditions and are substantially different from intact neuronal networks. Therefore, a number of important questions in cellular neurophysiology can only be studied in intact or semi-intact preparations.

3.3.1.2  Invertebrate Ganglia

Extending the same approach to in situ conditions has proven ­difficult and, hence, slow to develop. In in situ conditions, extracellular application of fluorescent dyes cannot provide single-cell resolution because of the large background fluorescence from the dye bound indiscriminately to all membranes in the preparation. Thus, in the first attempt to investigate regional electrical ­properties of individual neurons in situ in the barnacle supraesophageal ganglion, Ross and Krauthamer (1984) used transmission (absorption) measurements and, as in the experiments on neurons in culture, bath applied voltage-sensitive dyes to record optical signals from processes of individual nerve cells. Because the S/N of these measurements was not nearly as good as in recordings from monolayer culture, extensive averaging (approximately 300 trials) was required to obtain signals from processes of single neurons. The relatively small signal size that requires extensive averaging limits the utility of this approach for studying synaptic interactions and plasticity. Also, when many neurons are active in a densely packed neuropile it is difficult to determine the source of the signal if all the cells and processes are stained by extracellular dye application (Konnerth et al. 1987). Thus, the prospect for using measurement of voltagesensitive dye absorption or fluorescence with extracellular staining is limited, and no further experiments requiring subcellular resolution have been reported using this type of staining. The limits to the sensitivity in these measurements were determined primarily by (a) the relatively low voltage sensitivity of the available dyes; and (b) the large background light intensity inherent to both absorption and fluorescence measurements with extracellular application of the dye, which translates into low fractional change in light intensity (DI/I) related to Vm transients (Waggoner and Grinvald 1977).

3.3.2 Intracellular Application of Dyes 3.3.2.1  Invertebrate Ganglia

A different approach to optical analysis of electrical events in the processes of individual nerve cells is to stain particular neurons in situ selectively by intracellular application of an impermeant flu3.3.1.1  Dissociated Neurons in Culture In 1981 Grinvald, Ross, and Farber demonstrated that voltage-­ orescent voltage-sensitive dye. This approach is based on pioneersensitive dyes and multisite optical measurements can be employed ing measurements, carried out on the giant axon of the squid, which successfully to determine conduction velocity, space constants, and demonstrated that optical signals may be obtained when the dye is regional variations in the electrical properties of neuronal processes applied from the inside (Davila et  al. 1974; Cohen et  al. 1974;

3.3.1 Extracellular Application of Dyes

Imaging Submillisecond Membrane Potential Changes from Individual Regions of Single Axons, Dendrites and Spines    Chapter 3 ●

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FIGURE 3.1.  Transmission (A) and fluorescence (B) signals recorded with a single photodiode from extracellularly stained neuroblastoma cells in culture. Multiple site recording using a photodiode array is shown on the right. Adapted from Grinvald et al. (1981).

Salzberg 1978; Gupta et  al. 1981). In these experiments, optical signals were obtained from the large membrane surface area of the giant axon. Additional efforts were required to demonstrate that the same approach is feasible at the spatial scale of normal size neurons and their processes. The initial experiments using intracellular application of the voltage-sensitive dyes were carried out by Obaid et  al. (1982) and Grinvald et  al. (1987) on leech neurons. The results demonstrated the essential advantages of using intracellular application of fluorescent potentiometric probes. Fluorescence measurements are more effective than absorption measurements when measuring from a small membrane area (Waggoner and Grinvald 1977), particularly in situations where the image of the object (e.g., thin process) is much smaller than the size of the ­photodetector picture element (pixel) (Grinvald et al. 1982; Cohen and Lesher 1986). When transmitted light is used, only a small fraction of the total light captured by individual pixels will be ­modulated by the signal from the neuronal process (dendrite or axon) projected onto that pixel (DI). Most of the light will be ­projected directly and will only contribute to the resting light intensity (I ). Thus, the fractional signal (DI/I) will be very small. On the other hand, in fluorescence measurements, practically all of the light ­projected onto individual pixels will come from the object (if autofluorescence is negligible) regardless of the fraction of the pixel surface area covered by the image of the object. In the experiments reported by Grinvald et  al. (1987), fluorescence measurements were used to record APs and synaptic potential signals from processes of selectively stained single neurons of the leech (Fig. 3.2). As predicted, the S/N was substantially improved relative to prior absorption measurements, but it was still too low to be of practical value – the available sensitivity was insufficient for ­multiple site optical recording of complex electrical interactions at the level of

thin neuronal processes. Due to the low sensitivity of the measurements, elaborate correction procedures and extensive temporal averaging were necessary to improve the S/N. Additionally, spatial resolution was sacrificed and the signals reported were recorded from one location with a single photodiode. Moreover, the signals originated from a relatively large region of neuropile containing many processes, albeit from a single identified neuron (Fig. 3.2C). Despite low sensitivity, these experiments clearly showed the advantages of selective staining of individual neurons by intracellular application of the dye. The limits to the sensitivity of these measurements were determined primarily by (a) the relatively low voltage sensitivity of the available dyes (amino-phenyl styryl dyes, e.g., RH437 and RH461; fractional change in fluorescence intensity per AP, in intracellular application, of the order of 0.01–0.1%); (b) the choice of the excitation light bandwidth; and (c) the relatively low intensity of the incident light that could be obtained from a 100-W mercury arc lamp. At the time, the reported sensitivity was the result of a modest screening effort suggesting that better signals might be obtained by (a) synthesizing and screening new molecules for higher ­sensitivity; (b) increasing the concentration of the dye to increase the fluorescence intensity; (c) using an excitation light source capable of providing higher excitation light intensity and better stability; and (d) using detector devices with lower dark noise and adequate spatial and ­temporal resolution. Following this rationale, the first substantial improvement in the S/N was obtained by finding an intracel­lular voltage-sensitive dye with sensitivity in intracellular application two orders of magnitude higher than what was previously available (the amino-naphthalene styryl dye JPW1114 synthesized by J. P. Wuskell and L. M. Loew at the University of Connecticut Health Center; DF/F per AP, in intracellular application, of the order of 1–10%;

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Chapter 3    M. Canepari et al. ●

1700; Opti-Quip, Highland Mills, NY)]. The dramatic improvement in sensitivity and the use of 12 × 12 or 24 × 24 element photodiode detectors array (Centronix, Inc., Newbury Park, CA) allowed simultaneous multiple sites monitoring of electrical signals from processes of invertebrate neurons in isolated ganglia (Antic and Zecevic 1995; Zecevic 1996; Antic et al. 2000). Figure 3.3 shows a diagram of the optical arrangement for ­epi-fluorescence measurement and a typical multiple site recording from axo-­dendritic processes of an individual neuron from the land snail Helix aspersa. This type of recording permitted the analysis of the pattern of initiation and propagation of spikes in neuronal processes and provided direct information about the location and the number of spike trigger zones in a particular nerve cell. Also, it was ­possible to monitor directly spike propagation failure at axonal branch points (Antic et al. 2000). In all experiments on invertebrate ­neurons, individual nerve cells in isolated ganglia were observed using wide-field transmission microscopy and selectively stained intracellularly with the voltage-sensitive dye by iontophoresis from beveled sharp electrodes (Obaid et al. 1982; Grinvald et al. 1987; Antic and Zecevic 1995). The substantial improvement in the sensitivity of optical recording was accompanied by new demands for increased spatial and temporal resolution. Increased resolution, in turn, tended to diminish the S/N. The invertebrate neuron studies described above required spatial and temporal resolution that could only be achieved by using substantial temporal averaging (from 4 to 100 trials) to improve the S/N. In addition, the spatial resolution was limited by the available S/N to about 20–50 mm long sections of axo-dendritic processes. Clearly, further improvements were needed in order to reduce the temporal averaging and increase spatial resolution. The limits to the sensitivity in these measurements were determined primarily by (a) the sensitivity of the available voltage dyes; (b) the choice of the excitation bandwidth; (c) the maximum excitation light intensity that can be obtained from a 250-W xenon arc lamp; and (d) the dark noise of the photodiode array which was the dominant noise at the fluorescence intensities recorded from individual neurons stained by intracellular application of the dye.

3.3.2.2  Vertebrate Brain Slices

FIGURE 3.2.  Single photodiode optical recording from a neuron selectively stained by intracellular application of a voltage-sensitive dye. (A) Leech motoneuron following a 20-min iontophoretic injection with the styryl dye, RH-461. The soma, main process, and its arborization within the neuropile are all clearly visible. (B) Optical recording of the AP in 175-mm long axonal segment of a P sensory neuron in the anterior root. Twelve trials were averaged. (C) Optical recording of the excitatory postsynaptic potential in the arborization of the L-motor neuron. The experimental arrangement for interganglionic stimulation and recording shown on top. Microelectrode recordings of the presynaptic AP in the P sensory neuron and the postsynaptic potential in the soma of the L motor neuron are shown. The region of the neuropile that was monitored is shown in the inset. The optical recording of the postsynaptic potential from the neuropile in the adjacent ganglion is shown at the bottom. Adapted from Grinvald et al. (1987).

Antic and Zecevic 1995). The higher sensitivity of this dye translates directly into higher S/N. At this stage, the sensitivity was also improved by using a more powerful excitation light source [Osram, XBO 250W OFR powered by a low-noise power supply (Model

It was of considerable interest to apply Vm-imaging techniques to the dendrites of vertebrate CNS neurons in brain slices. This possibility was first demonstrated with work on layer 5 neocortical pyramidal neurons (Antic et  al. 1999) that established a basic protocol for intracellular labeling of individual nerve cells in slices and described the method for simultaneous optical recording of electrical signals from multiple sites on apical, oblique, and basal dendrites. In these experiments, individual pyramidal neurons in slices were observed using infrared dark-field video microscopy and stained selectively by intracellular application of the voltage-sensitive dye by diffusion from a patch electrode in a whole-cell configuration. The major problem in injecting vertebrate neurons from patch pipettes was leakage of the dye from the electrode into the extracellular medium before the electrode is attached to the neuron. Patching requires pressure to be applied to the electrode during electrode positioning and micromanipulation through the tissue. This pressure ejects solution from the electrode. To avoid extracellular deposition of the dye that binds to the slice and produces large background fluorescence, the tip of the electrode was filled with dye-free solution, and the electrode was backfilled with dye solution. The amount of dyefree solution in the electrode tip and the applied pressure has to be adjusted empirically to ensure that no dye leaks from the electrode before the seal is formed. Usually, no pressure is applied before the electrode enters the slice. Low pressure (~30 mbar) is used

Imaging Submillisecond Membrane Potential Changes from Individual Regions of Single Axons, Dendrites and Spines    Chapter 3 ●

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FIGURE 3.3.  Multiple site optical recording with a photodiode array from a neuron selectively stained by intracellular application of a voltage-sensitive dye. (A) Schematic drawing of the optical apparatus for multiple site epi-fluorescence measurements. The photodiode array was placed at the plane where the objective makes a real, magnified, and inverted image. Epi-illumination was provided via a 10×, 0.4 NA, long working distance objective. The preparation was illuminated with the green portion (interference filter 520 ± 45 nm) of the output of a 100-W xenon arc lamp. Fluorescence emission from the preparation was selected by means of a dichroic mirror and an appropriate barrier filter (Schott RG610). The output of each detector was amplified individually, filtered, multiplexed, digitized, and stored in a computer also used to analyze and display the data. (B) Optical recordings of AP signals from elements of a photodiode array positioned over the image of the axonal arborization of a Helix metacerebral cell from the left cerebral ganglion. The neuron was injected with the voltage-sensitive dye JPW1114 (structural formula shown in lower right panel). Spikes were evoked by transmembrane current pulses delivered through the recording microelectrode in the soma. The cell was depolarized from the resting Vm of –64 mV. Each optical trace represents 70 ms of recording centered on the peak of the spike. The recordings are shown divided by the resting light intensity. The arrow indicates the direction and the stated value of the relative fluorescence change (DF/F). Each diode received light from a 50 mm × 50 mm area in the object plane. A total of 90 trials were averaged to improve the S/N from distal processes. Optical signals were found in the regions of the array that correspond closely to the geometry of the cell. Data from the detectors that do not receive light from the cell were deleted to improve clarity and allow color-coded display, shown in (D), based on a relative scale applied separately to each trace. (C) AP signals from four different locations indicated in (B) scaled to the same height are compared on an expanded time scale to determine the site of the origin of the AP and the direction of propagation. (D) Color-coded representation of the data shown in (B) indicating the size and location of the primary spike trigger zone and the pattern of spike propagation. Consecutive frames represent data points that are 1.6 ms apart. Color scale is in relative units with the peak of the AP shown in red. Adapted from Zecevic (1996).

during electrode positioning in the slice, and the final pressure of ~100 mbar, necessary for cleaning the surface membrane of the cell, is applied immediately (10 s) before the seal formation. Using this method, it was possible to load neurons routinely without increasing the background fluorescence of the surrounding tissue. At the end of a ~1-h diffusion period, the patch pipette was detached and the injected dye was allowed to spread for an additional 2 h before the start of optical recording. Within this time, dye would reach the apical tuft branches in layer 1, ~1 mm from the soma. The results of these experiments demonstrated (a) that loading vertebrate neurons with a voltage-sensitive dye using patch electrodes was possible without contamination of the extracellular environment; (b) that brain slices did not show significant autofluorescence at the excitation/emission wavelengths used [in contrast with the experience of Grinvald et al. (1987)]; (c) that pharmacological effects of the dye were ­completely reversible; (d) that the level of photodynamic damage was low enough to permit meaningful measurements and could be reduced further; and (e) that the sensitivity of optical recording with a 464-element photodiode array was comparable to that reported for invertebrate neurons. At this level of sensitivity, however, modest temporal averaging in recording AP signals was still required, and spatial resolution was limited by the available S/N to approximately 20-mm long sections of dendritic processes

(Antic et al. 1999). The limits to the S/N in these initial measurements from vertebrate neurons were determined by the same factors as described above for invertebrate preparations.

3.3.2.3  I mprovement in Sensitivity with CCD Cameras

The second significant improvement in recording sensitivity in measurements from individual nerve cells (after the synthesis of a dye far more suitable for intracellular application) was the introduction of a cooled, back-illuminated CCD camera in place of the 464-element diode array. Fluorescence measurements from individual neurons have a lower range of intensities (with correspondingly lower absolute amplitude of the shot noise) so that the dominant noise, in recordings with a 464-diode array, was the relatively large dark noise of the amplifier-photodiode circuit. Thus, the dark noise of the photodiode array limited the detection system sensitivity, rendering it significantly lower than the theoretically attainable shot noise limit. It was then realized that in this range of illumination intensities (from 5 × 103 to 5 × 106 photons/ms) the CCD camera can approach the ideal (shot noise limited) sensitivity (Wu et  al. 1999; see also Chap. 1). The back-illuminated CCD camera, because it is cooled and can have about 1,000 times smaller

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FIGURE 3.4.  Optical detectors for voltage-sensitive dye recordings. (A) The performance of a 464-element photodiode array and a cooled back-illuminated CCD camera are compared directly using the same preparation. The measurements were done at a relatively low resting light level, comparable to the resting fluorescence intensity in recordings from processes of selectively stained individual neurons. Upper trace: recording of a step change in light intensity adjusted in amplitude to be at the limit of resolution (S/N » 1) for the ­photodiode array. Lower trace: the recording of the same signals using the high-speed, cooled CCD camera. The improvement in the S/N ratio by a factor of approximately 20 is attributable to the large reduction in dark noise. (B) AP signals in distal segments of basal dendrites of a pyramidal neuron obtained with a cooled CCD camera. A composite fluorescence image of a layer 5 pyramidal neuron in recording position shown on the left. Optical signals from distal dendritic segments at distances larger than 180 mm, as indicated, shown on the right. Each trace is the product of temporal (four trials) and spatial (6–9 pixels) averaging. Adapted from Antic (2003).

pixel surface area and proportionally smaller dark currents compared to available diode arrays, exhibits substantially lower dark noise. Test measurements performed at low light level (Wu, Cohen, and Zecevic, unpublished observations) established that a particular cooled CCD camera (FastOne, Pixel Vision Inc., Beaverton, OR) improved the sensitivity by a factor of approximately 20 (Fig. 3.4A). The upper trace in Fig. 3.4A shows the recording of a step change in light intensity adjusted in amplitude to be at the limit of resolution (S/N = 1) for the photodiode array. The lower trace is the recording of the same steps using the high-speed, cooled CCD camera. This result is a demonstration of the remarkable improvement in S/N resulting from the large reduction in dark noise. The increase in sensitivity with the introduction of advanced CCD cameras allowed, for the first time, recording of the spatial and temporal dynamics of electrical events in thin, distal dendritic processes

that cannot be probed with conventional patch-electrode techniques. The optical approach made possible simultaneous measurements of the Vm transients in basal and oblique dendrites of pyramidal neurons during single APs, trains of APs (Fig. 3.4; Antic 2003), as well as the analysis of synaptic potential initiation and spread in terminal dendrites of the glomerular tuft of individual mitral cells in the olfactory bulb slice (Djurisic et al. 2004). The optical measurements of Antic (2003) provided unique evidence that, in contrast to apical dendrites, basal and oblique dendritic processes impose modest amplitude and time course modulation on backpropagating APs and are robustly invaded by the somatic spike even when somatic firing rates reach 40 Hz (Fig. 3.4B). A compartmental model incorporating AP peak latencies and half-widths obtained from optical measurements indicated that the specific intracellular resistance (Ri) is less than 100 W cm. The data obtained simultaneously from multiple dendritic sites and ­subsequently linked to a numerical simulation, revealed that all synaptic locations along basal and oblique dendrites, situated within 200 mm of the soma, experience strong and near-simultaneous (latency 100 trials), as required for monitoring fast axonal APs at physiological temperature. In addition, it was still not possible to monitor Vm signals at the higher optical magnification and finer spatial scale necessary for resolving electrical events at the level of individual dendritic spines. Clearly, further improvements in sensitivity were needed.

3.3.2.4  I mprovements in Sensitivity with Monochromatic, Laser Light Excitation

A possible approach toward achieving high-speed multiple-site optical recordings of membrane voltage from axons and axonal collaterals as well as from individual dendritic spines in brain slices is wide-field epi-fluorescence microscopy applied at high optical magnification. Several alternative optical approaches to Vm imaging are also available; these will be discussed in Sect. 3.3.2.5. The limited sensitivity of available epi-fluorescence Vmimaging methods required extensive averaging (>100 trials) in

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FIGURE 3.6.  Spatial distribution of subthreshold EPSP signals is uniform in thin terminal dendritic branches of a mitral cell dendritic tuft. (A, B) Fluorescence image of a mitral cell tuft obtained with a conventional high-resolution CCD (A) and with a fast, low-resolution (80 × 80 pixels) CCD used for voltage imaging (B). Six color-coded recording locations indicated on the image in (B) correspond to traces shown in (C). (C) Trace display of Vm signals. Traces are spatial averages of colored pixels. Bottom black traces, electrical recordings from soma. Left traces: single-trial recordings of the small EPSP signal are at the limit of amplitude resolution. Middle traces: Nine averages of the small EPSP signals shown on the left improved the S/N ratio. Right traces: nine averages of the EPSP four times larger in amplitude. Adapted from Djurisic et al. (2008).

recording fast axonal AP signals at high frame rates (Palmer and Stuart 2006). Similarly, low sensitivity was obtained at an order of magnitude higher optical magnification required to resolve Vm signals from individual dendritic spines (Palmer and Stuart 2009). Because extensive averaging (>100 trials) makes most physiologically relevant experiments very difficult or impossible, a substantial improvement, by a factor of 10–100, in the sensitivity was desirable. Within the constraints imposed by (3.1), one way to increase the S/N with a given voltage-sensitive dye is to increase the photon flux (F) by increasing the excitation light intensity. Another possibility is to increase the fractional fluorescence change per unit change in Vm (sensitivity of the dye) by choosing the optimal ­excitation wavelength. Following this rationale, Holthoff et  al. (2010) used one of the most sensitive voltage-sensitive dyes in terms of S/N (JPW3028; Djurisic et al. 2004; Zhou et al. 2008; Acker and Antic 2009), and improved both the excitation (and, hence, emission) light intensity and the relative fluorescence change in response to Vm change by utilizing a laser as an illumination source in wide-field epi-fluorescence microscopy mode. In measurements from layer 5 pyramidal neurons in rat visual cortex slices, the light from a frequency-doubled 200-mW diodepumped Nd:YVO4 continuous wave laser emitting at 532 nm (Excelsior 532 single mode; Newport-Spectra-Physics, Mountain

View, CA) was directed to a quartz optical fiber (TILL Photonics GmbH, Gräfelfing, Germany) coupled to the microscope via a single-port epi-fluorescence condenser (TILL Photonics) designed to overfill the back aperture of the objective. In this way, approximately uniform illumination of the object plane was attained. Figure 3.7 depicts the experimental setup schematically. The fractional amplitude noise in low-noise solid-state lasers (100 trials) was required to obtain useful information. This amount of averaging makes experiments difficult and prone to errors. Clearly, a significant improvement in sensitivity brought about by laser excitation will facilitate further studies. A typical optical recording (average of four trials) from the axon of a layer 5 pyramidal neuron from the visual cortex, shown in Fig. 3.9B– E, illustrates the advantages of this imaging approach. The fluorescence image of a part of the axonal arbor in the recording position is shown in panel (A) together with the anatomical reconstruction of the stained neuron from a z-stack of two-photon images. Panel (C) illustrates optically recorded local AP signals from the five axonal regions indicated on the CCD image. From this type of data, it was possible to characterize the pattern of spike initiation and propagation in the main axon and its collaterals as well as the waveform of signals from different locations (panel D). The color-coded representation of the same data shows the spatiotemporal distribution of AP signals (panel E). These measurements established that the superior sensitivity of wide-field laser-excitation epi-fluorescence recordings permit Vm imaging from the axonal arbor, including small diameter axon collaterals, with high temporal and spatial resolution.

Improved Vm Imaging from Dendritic Spines

The sensitivity of recording from dendrites and axons at relatively low optical magnification described above indicated that it should be possible to increase the optical magnification by a factor of 10 and monitor Vm transients from individual dendritic spines and, potentially, from presynaptic axonal varicosities/boutons. Recording electrical events from individual dendritic spines is important for several reasons. Spines are likely to play a critical role in the input–output transform carried out by an individual neuron. They receive most of the excitatory synapses in many brain regions and may serve as calcium compartments, which appear to be necessary for input-specific synaptic plasticity (Yuste and Denk 1995; Sabatini et  al. 2002; Noguchi et al. 2005). In the last decade, a number of investigators (e.g., Winfried Denk, Rafael Yuste, Karel Svoboda, Bernardo Sabatini, and Haruo Kasai) revisited previously well-articulated questions and ideas, and analyzed open problems in spine physiology with the aid of experimental measurements. The methodology was developed to measure Ca2+ signals from ­individual spines with great precision using two-photon microscopy, and many aspects of spine physiology have been illuminated. The electrical behavior of spines, however, is less well understood and controversial. For a long time, the role of spines had to be considered on purely theoretical grounds because it was technically impossible to measure Vm signals from individual spines. Theoretical work (Jack et al. 1975; Segev and Rall 1988) and, more recently, several experimental studies (Bloodgood and Sabatini 2005; Noguchi et al. 2005; Araya et al. 2006a, b) provided indirect evidence that the

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electrical characteristics of dendritic spines might have important implications for integrative function and for the plastic properties of nerve cells. Other studies based on diffusion measurements (Svoboda et al. 1996) as well as on multicompartmental modeling (Koch and Zador 1993) indicated that spines may not play a significant electrical role. This question is still unresolved because, with one notable exception (Palmer and Stuart 2009; see below), it has not been ­possible to document directly the electrical behavior of dendritic

spines, owing to the limited sensitivity of the available measurement ­techniques at the requisite spatial resolution. Thus, a critical challenge, both conceptually and technically, was to develop an approach for the direct analysis of Vm signals at the spatial scale of individual dendritic spines. The improvement in sensitivity described above made this type of recording possible (Holthoff et al. 2010) as illustrated in Fig. 3.10. In these measurements, a magnified image of a spiny dendrite of a stained neuron was projected onto a CCD camera

FIGURE 3.10.  Backpropagated AP signals from individual dendritic spines and parent dendrites. (A, B) Left panels: Upper micrographs – fluorescence images of dendritic spiny branches obtained with the CCD camera for voltage imaging. Lower micrographs – anatomical reconstructions obtained from deconvoluted stacks of spinning-disk confocal images. Right panels: Fluorescence intensity traces from locations 1 to 3 outlined on CCD images. Single-trial recordings and temporal averages of four and nine trials are shown. Bottom traces: electrode recordings from the soma. The bAP signals are absent from regions without spines (locations 2 and 3). (C) A fluorescence image of a section of a spiny dendrite. (D) Anatomical reconstruction from a stack of confocal images. (E) Comparison of bAP waveform in the spines and parent dendrite. (F) Summary results: comparison of bAP duration in the spines and parent dendrites. The individual results of waveform comparison from 20 neurons are shown together with the average values ± SEM. Adapted from Holthoff et al. (2010).

Imaging Submillisecond Membrane Potential Changes from Individual Regions of Single Axons, Dendrites and Spines    Chapter 3 ●

and fluorescence intensity changes were monitored from multiple locations. The bAP-related signals from dendritic spines were clearly resolved in single-trial measurements and the S/N was further improved by averaging a small number of trials (4–9). One concern with respect to these measurements was that scattered light from the parent dendrite, in wide-field fluorescence measurements, might contaminate signals from individual spines. To address this issue, Holthoff et  al. (2010) compared dendritic signals from individual spines with recordings from analogous regions without spines and found that the signals from regions without spines were smaller than the noise in the measurement (Fig. 3.10A, B, locations 2 and 3). Thus, the interference from scattered light was insignificant in the superficial layers of the slice. Another concern was the amount of photodynamic damage caused by the high-intensity excitation light. In several experiments, the very first single-trial optical recording of the bAP (control signal) was compared with the last bAP signal at the end of the experiment. The results showed that, in the range tested (up to 25 recording trials) the first and the last bAP signal were identical, indicating that the photodynamic damage was not significant. From the type of data shown in Fig. 3.10, it was possible, for the first time, to determine and compare the time course of the bAPs signals in spines and the parent dendrites (Holthoff et  al. 2010). The waveforms of the bAP signals were reconstructed from the data obtained at 2-kHz frame rate using cubic spline interpolation and compared on an expanded time scale. Figure 3.10E shows that both the upstroke and the downstroke of the AP in spines and dendrites closely overlapped. The summary result from 20 different neurons (Fig. 3.10F) showed that signals from spines and dendrites did not differ significantly. Thus, these results demonstrate that bAPs in spines have a rapid time course that is very similar to that of spikes recorded in the parent dendrite. The rapid time course of the bAP in the spines may be a critical determinant for the precise regulation of spike timingdependent synaptic plasticity within a very narrow time window (Caporale and Dan 2008).

3.3.2.5  Alternative Approaches

A possible way to further improve Vm imaging from individual ­neurons is by synthesizing new organic probes with characteristics designed specifically for a particular application. In recent studies, Leslie Loew, Srdjan Antic, and their collaborators introduced a new series of long-wavelength voltage-sensitive dyes for intracellular application (Wuskell et al. 2006; Zhou et al. 2007). These new voltage probes (called blue dyes; Shoham et al. 1999) extended the range of excitation wavelengths to near 700 nm, with emission reaching 800–900 nm. Longer wavelength dyes permit deeper penetration by the excitation light into the nervous tissue which could be of considerable importance in optical measurements for both brain slice and in vivo preparations (see Chap. 2). These dyes also offer new possibilities for the design of combined recordings with multiple indicators (Canepari et al. 2008; Chap. 4). The new probes seem to be characterized by somewhat higher voltage sensitivity in terms of the fractional fluorescence change in response to a unit change in Vm, when applied intracellularly (but see Fig. 3.8C and the discussion of dye sensitivity in Sect. 3.3.2.4). The sensitivity of recording from individual neurons in terms of the S/N, however, has not, as yet, been fully exploited with the application of blue dyes (Zhou et al. 2007, 2008). Additional sensitivity comparisons remain to be carried out at similar excitation light intensities and for each particular preparation. Both the new long-wavelength voltage-sensitive dyes and the more conventional probes excited by the green portion of the visible

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spectrum were used, together with stationary, small spot laser ­illumination, to investigate the dynamics of backpropagated APs in the terminal sections of basal dendrites (dendritic tips) of prefrontal cortical pyramidal neurons, a region that has never been probed for  electrical signals by any method (Zhou et  al. 2008). In this recording mode, the excitation light was provided from laser sources emitting at 532, 633, or 658 nm. The laser beam was directed into one end of a 0.2-mm diameter optical fiber (light guide) with a collimator attached to the other end of the fiber which was coupled to the epi-fluorescence port of a microscope. In this way, a stationary spot of laser light (25–50 µm in diameter) was projected onto the object plane. For recordings of voltage-sensitive dye signals, a selected region of the neuronal processes of interest was moved into the laser spot using the X–Y microscope positioning stage. The sensitivity of these measurements in recordings from 25 to 50 µm long sections on dendritic processes allowed optical monitoring of AP signals from the tips of basal dendrites, as well as from individual axons, with minimal temporal averaging. Representative AP-related signals obtained in these studies are shown in Figs. 3.11 and 3.12. The results indicated that in short (100 trials) as well as adding averaged results to create so-called super averages was required to extract useful information. The necessity for extensive averaging makes repeated recordings under different conditions difficult or impossible. In addition, signal averaging limits this approach to studies of relatively simple phenomena while many aspects of dendritic signal integration are too complex to be analyzed by averaging. The second disadvantage of confocal measurements is that the recording speed is restricted to approximately 800 Hz by the available S/N. This sampling rate is insufficient for correct reconstruction of AP and EPSP signal size and shape according to the Nyquist–Shannon sampling theorem (Roberts 2004). The theorem defines the Nyquist rate, the minimum sampling rate required to avoid distortion of the analog signal, to be equal to twice the highest frequency contained within the signal. The upstroke of the AP waveform (threshold to peak) at room temperature is completed in 300–600 µs corresponding to frequencies of 1.6–3 kHz and, thus, requiring optical recording frame rates in the range of 3–6 kHz. At physiological temperature this requirement is even more stringent. It is helpful that the general shapes of the AP and EPSP waveforms are well defined from electrical measurement, so that aliasing, a certain type of unwanted distortion of the signal, can be safely excluded. This fact allows a modest relaxation of the strict Nyquist rate rule. Nevertheless, the data obtained with sampling rates significantly lower than the Nyquist rate must be regarded as approximate because of the significant distortion of both the waveform and the amplitude of the signal. Finally, the third important limitation of laser-scanning confocal Vm imaging is that, in applications where the goal is to monitor electrical events at many locations on an individual neuron, a line scan imaging mode cannot replace a true multiple site optical recording. Notwithstanding these limitations, the results described in Palmer and Stuart (2009) argue convincingly that bAPs invade dendritic spines without significant voltage loss. Additionally, the measurements of EPSP signals at different Vm levels showed that voltage-activated channels do not significantly boost the voltage response in dendritic spines during synaptic input. This work also emphasizes the crucial importance of adequate spatial resolution in recordings from small structures within an opaque brain slice tissue. If the recording is carried out from neurons deep in a slice, the required resolution can only be provided by microscopy modes that reject scattered and out of focus light (confocal microscopy, SHG microscopy, and two-­photon fluorescence microscopy). The same limitation in sensitivity applies to SHG imaging of Vm signals, a nonlinear optical technique that generates a similar or lower photon flux compared to two-photon imaging (Dombeck et al. 2005). There are two important additional restrictions to SHG imaging. First, SHG is, essentially, a nonlinear scattering pheno­ menon and, as such, is predominantly in the forward direction (see Chaps. 11 and 13), limiting this approach to thin preparations and precluding epi-illumination. Secondly, SHG requires a noncentro-symmetrical radiating (scattering) source. If the molecular distribution of the probe in a focal volume is partially symmetrical, with molecules oriented in opposing directions, they will produce SHG signals with opposing phases. This leads to a partial destructive interference of SHG signals, reducing the signal size by a factor that is unknown in the general case (Moreaux et al. 2000). Thus, the same Vm transient in different dendritic compartments may produce optical

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FIGURE 3.12.  Optical recording of AP signals from the axon using a longwavelength (blue) voltage-sensitive dye JPW-4090. (A) Composite fluorescence image of a neuron; axon in recording position. Boxes mark two regions of interest (ROI) along the axon. Single-trial (no averaging) VSD recording from ROI 1 is aligned with the somatic whole-cell recording (soma). (B) AP signals from axonal segment 85 mm away from the cell body (ROI 1; average of six trials) and from a more distal (185 mm) region (ROI 2; average of nine trials). Adapted from Zhou et al. (2007).

signals of dramatically different amplitudes, preventing correct calibration of the optical response in terms of Vm from measurements at any one site. The same difficulty in calibrating optical signals on an absolute scale exist in Vm imaging using intracellular voltagesensitive dyes, as described above, albeit for a different reason. The first attempt to analyze electrical events in individual spines (Nuriya et al. 2006) was based on recording SHG signals. The results of this work provided excellent impetus for making Vm imaging with single-spine resolution possible. The same results, however, are a good illustration of the current methodological difficulties. The low sensitivity of these measurements required extensive averaging (>100 trials) but still resulted in an insufficient S/N, precluding full analysis of the signal size and shape. It is noteworthy that signal averaging has strict limitations and a further improvement in the S/N by a factor of only two would require averaging more than 1,000 trials, which is not feasible in most experiments. Thus, no further application of this approach to monitoring electrical events from individual spines has been reported. Finally, the most modern approach to Vm imaging is a methodology focused on genetically encoded protein Vm sensors (see Chap. 14 for more details). The genetic approach could potentially develop into an ideal method to selectively label and monitor individual classes of neurons. Thus, protein sensors (voltage-sensitive fluorescent proteins) are being extensively investigated (Siegel and Isacoff 1997; Sakai et al. 2001; Ataka and Pieribone 2002; Chanda et al. 2005; Knöpfel et al. 2003; Dimitrov et al. 2007) and some of the prototype molecules have shown considerable promise in their ability to detect Vm transients (see Chap. 14 for more details). At present, however, this approach to monitoring Vm signaling in individual neurons is limited in several ways. First, the sensitivity and the response time of genetically encoded protein sensors are, as yet, insufficient. In addition, the problems regarding linearity of response, adequate expression in vertebrate neurons, and probable undesirable effects (e.g., significant capacitive load) on neuronal physiology have not been resolved. Thus, the practical application of this approach must await further development.

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FIGURE 3.13.  Imaging Vm in dendritic spines using confocal line scan microscopy mode. (A) A fluorescence image of a basal dendrite with two dendritic spines. Scale bar = 1 mm. Dashed red line indicates a laser line-scan traversing two spines and the resulting average fluorescence change in response to ~100 bAPs (right). Red arrowhead indicates timing of stimulus, and colored bars represent four labeled regions of interest. (B) Fluorescence traces (colored) in response to somatically evoked bAPs (black) for the regions of interest shown in (A). Adapted from Palmer and Stuart (2009).

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Chanda B, Blunck R et  al (2005) A hybrid approach to measuring electrical activity in genetically specified neurons. Nat Neurosci 8:1619–1626. Cohen LB, Lesher S (1986) Optical monitoring of membrane potential: methods of multisite optical measurement. Soc Gen Physiol Ser 40:71–99. Cohen LB, Salzberg BM et  al (1974) Changes in axon fluorescence during activity: molecular probes of membrane potential. J Membr Biol 19:1–36. Dainty JC (1984) Laser speckle and related phenomena. Springer, New York. Davila HV, Cohen LB, Salzberg BM, Shrivastav BB (1974) Changes in ANS and TNS fluorescence in giant axons from Loligo. J Membr Biol 15:29–46. Dimitrov D, He Y et al (2007) Engineering and characterization of an enhanced fluorescent protein voltage sensor. PLoS ONE 2:e440. Djurisic M, Antic S, Chen WR, Zecevic D (2004) Voltage imaging from dendrites of mitral cells: EPSP attenuation and spike trigger zones. J Neurosci 24:6703–6714. Djurisic M, Popovic M, Carnevale N, Zecevic D (2008) Functional structure of the mitral cell dendritic tuft in the rat olfactory bulb. J Neurosci 28:4057–4068. Dombeck DA, Sacconi L, Blanchard-Desce M, Webb WW (2005) Optical recording of fast neuronal membrane potential transients in acute mammalian brain slices by second-harmonic generation microscopy. J Neurophysiol 94:3628–3636. Fisher JA, Barchi JR et  al (2008) Two-photon excitation of potentiometric probes enables optical recording of action potentials from mammalian nerve terminals in situ. J Neurophysiol 99:1545–1553. Gonzalez JE, Tsien RY (1995) Voltage sensing by fluorescence resonance energy transfer in single cells. Biophys J 69:1272–1280. Gonzalez JE, Tsien RY (1997) Improved indicators of cell membrane potential that use fluorescence resonance energy transfer. Chem Biol 4: 269–277. Gratton E, vande Ven MJ (1989) Laser sources for confocal microscopy. In Pawley JB (ed) Handbook of biological confocal microscopy. Springer, New York. Grinvald A, Farber IC (1981) Optical recording of calcium action potentials from growth cones of cultured neurons with a laser microbeam. Science 212:1164–1167. Grinvald A, Ross WN, Farber I (1981) Simultaneous optical measurements of electrical activity from multiple sites on processes of cultured neurons. Proc Natl Acad Sci U S A 78:3245–3249. Grinvald A, Hildesheim R, Farber IC, Anglister L (1982) Improved fluorescent probes for the measurement of rapid changes in membrane potential. Biophys J 39:301–308. Grinvald A, Salzberg BM, Lev-Ram V, Hildesheim, R (1987) Optical recording of synaptic potentials from processes of single neurons using intracellular potentiometric dyes. Biophys J 51:643–651.

Imaging Submillisecond Membrane Potential Changes from Individual Regions of Single Axons, Dendrites and Spines    Chapter 3 ●

Gupta, RK, Salzberg BM et  al (1981) Improvements in optical methods for measuring rapid changes in membrane potential. J Membr Biol 58: 123–137. Holthoff H, Zecevic D, Konnerth A (2010) Rapid time-course of action potentials in spines and remote dendrites of mouse visual cortex neurons. J Physiol 588:1085–1096. Iwasato T, Datwani A et  al (2000) Cortex-restricted disruption of NMDAR1 impairs neuronal patterns in the barrel cortex. Nature 406:726–731 Jack JJB, Noble D, Tsien RW (1975) Electric current flow in excitable cells. Oxford University Press, London. Kampa BM, Stuart GJ (2006) Calcium spikes in basal dendrites of layer 5 pyramidal neurons during action potential bursts. J Neurosci 26: 7424–7432. Kerr JN, Denk W (2008) Imaging in vivo: watching the brain in action. Nat Rev Neurosci 9:195–205. Knöpfel T, Tomita K, Shimazaki R, Sakai R (2003) Optical recordings of membrane potential using genetically targeted voltage-sensitive fluorescent proteins. Methods 30:42–48. Koch C, Zador A (1993) The function of dendritic spines: devices subserving biochemical rather than electrical compartmentalization. J Neurosci 14:4705–4715. Konnerth A, Obaid AL, Salzberg BM (1987) Optical recording of electrical activity from parallel fibres and other cell types in skate cerebellar slices in vitro. J Physiol 393:681–702. Kuhn B, Fromherz P, Denk W (2004) High sensitivity of Stark-shift voltagesensing dyes by one- or two-photon excitation near the red spectral edge. Biophys J 87:631–639. Larkum ME, Nevian T, Sandler M, Polsky A, Schiller J (2009) Synaptic integration in tuft dendrites of layer 5 pyramidal neurons: a new unifying principle. Science 325:756–760. Loew LM (1982) Design and characterization of electrochromic membrane probes. J Biochem Biophys Methods 6:243–260. Matsukawa H, Wolf AM, Matsushita S, Joho RH, Knöpfel T (2003) Motor dysfunction and altered synaptic transmission at the parallel fiber-Purkinje cell synapse in mice lacking potassium channels Kv3.1 and Kv3.3. J Neurosci 23:7677–7684. Milojkovic BA, Zhou WL, Antic SD (2007) Voltage and calcium transients in basal dendrites of the rat prefrontal cortex. J Physiol 585:447–468. Moreaux L, Sandre O, Mertz J (2000) Membrane imaging by second harmonic generation microscopy. J Opt Soc Am B 17:1685–1694. Nevian T, Larkum ME, Polsky A, Schiller J (2007) Properties of basal dendrites of layer 5 pyramidal neurons: a direct patch-clamp recording study. Nat Neurosci 10:206–214. Noguchi J, Matsuzaki M, Ellis-Davies GC, Kasai H (2005) Spine-neck geometry determines NMDA receptor-dependent Ca2+ signaling in dendrites. Neuron 46:609–622. Nuriya M, Jiang J, Nemet B, Eisenthal KB, Yuste R (2006) Imaging membrane potential in dendritic spines. Proc Natl Acad Sci U S A 103:786–790. Obaid AL, Shimizu H, Salzberg BM (1982) Intracellular staining with potentiometric dyes: optical signals from identified leech neurons and their processes. Biol Bull 163:388. Palmer LM, Stuart GJ (2006) Site of action potential initiation in layer 5 pyramidal neurons. J Neurosci 26:1854–1863. Palmer LM, Stuart GJ (2009) Membrane potential changes in dendritic spines during action potentials and synaptic input. J Neurosci 29:6897–6903. Parsons TD, Kleinfeld D Raccuia F, Salzberg BM (1989) Optical recording of the electrical activity of synaptically interacting neurons in culture using potentiometric probes. Biophys J 56:213–221.

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Parsons TD, Salzberg BM, Obaid AL, Raccuia-Behling F, Kleinfeld D (1991) Long-term optical recording of electrical activity in ensembles of cultured Aplysia neurons. J Neurophysiol 66:316–333. Roberts MJ (2004) Signals and systems: analysis using transform methods and MATLAB. McGraw-Hill, New York. Rohr S, Salzberg BM (1994) Multiple site optical recording of transmembrane voltage (MSORTV) in patterned growth heart cell cultures: assessing electrical behavior, with microsecond resolution, on a cellular and subcellular scale. Biophys J 67:1301–1315. Ross WN, Krauthamer V (1984) Optical measurements of potential changes in  axons and processes of neurons of a barnacle ganglion. J Neurosci 4:659–672. Sabatini BS, Oertner, TG, Svoboda, K. (2002) The life cycle of Ca2+ ions in dendritic spines. Neuron 33:439–452. Sakai R, Repunte-Canonigo V, Raj CD, Knopfel T (2001) Design and characterization of a DNA-encoded, voltage-sensitive fluorescent protein. Eur J Neurosci 13:2314–2318. Salzberg BM (1978) Optical signals from giant axon following perfusion or superfusion with potentiometric probes. Biol Bull 155:463–464 Segev I, Rall W (1988) Computational study of an excitable dendritic spine. J Neurophysiol 60:499–523. Shoham D, Glaser DE et al (1999) Imaging cortical dynamics at high spatial and temporal resolution with novel blue voltage-sensitive dyes. Neuron 24:791–802. Shu Y, Hasenstaub A, Duque A, Yu Y, McCormick DA (2006) Modulation of intracortical synaptic potentials by presynaptic somatic membrane potential. Nature 441:761–765. Siegel MS, Isacoff EY (1997) A genetically encoded optical probe of membrane voltage. Neuron 19:735–741. Stuart GJ, Sakmann B (1994) Active propagation of somatic action potentials into neocortical pyramidal cell dendrites. Nature 367:69–72. Stuart GJ, Dodt HU, Sakmann B (1993) Patch-clamp recordings from the soma and dendrites of neurons in brain slices using infrared video microscopy. Pflugers Arch 423:511–518. Svoboda K, Tank DW, Denk W (1996) Direct measurement of coupling between dendritic spines and shafts. Science 272:716–719. Vetter P, Roth A, Häusser M (2001) Propagation of action potentials in dendrites depends on dendritic morphology. J Neurophysiol 85:926–937. Waggoner AS, Grinvald A (1977) Mechanisms of rapid optical changes of potential sensitive dyes. Ann N Y Acad Sci 303:217–242. Wu JY, Cohen, LB, Falk CX (1999) Fast multisite optical measurement of membrane potential with two examples. In: Mason WT (ed) Fluorescence and luminescence probes. Academic Press, London. Wuskell JP, Boudreau D et al (2006) Synthesis, spectra, delivery and potentiometric responses of new styryl dyes with extended spectral ranges. J Neurosci Methods 151:200–215. Yuste R, Denk W (1995) Dendritic spines as basic functional units of neuronal integration. Nature 375:682–684. Zecevic D (1996) Multiple spike-initiation zones in single neurons revealed by voltage-sensitive dyes. Nature 381:322–325. Zhou W-L, Yan P, Wuskell JP, Loew LM, Antic SD (2007) Intracellular long-wavelength voltage-sensitive dyes for studying the dynamics of action potentials in axons and thin dendrites. J Neurosci Methods 164:225–239. Zhou WL, Yan P, Wuskell JP, Loew LM, Antic SD (2008) Dynamics of action potential backpropagation in basal dendrites of prefrontal cortical pyramidal neurons. Eur J Neurosci 27:923–936.

4

Combined Voltage and Calcium Imaging and Signal Calibration Marco Canepari, Peter Saggau, and Dejan Zecevic

4.1  Introduction This chapter addresses two important aspects of voltage imaging. The first aspect is the combination of membrane potential imaging (Vm imaging) with the optical measurement of intracellular Ca2+ transients. The correlation of a voltage recording with a measurement of another variable of biological interest is often necessary to unambiguously answer a specific question. Ca2+ signals are directly correlated with membrane potential when they originate from Ca2+ channels in the plasma membrane. This category includes voltagegated Ca2+ channels (VGCCs), glutamate receptors (in particular NMDA receptors) and other Ca2+ permeable pores. In all these cases, the charge influx associated with Ca2+ contributes, to a different extent, to the current underlying depolarization. In addition, the biophysical properties of several Ca2+ channels in the plasma membrane also depend on membrane depolarization. The two most important examples are VGCCs and NMDA receptors that unblock, in the presence of Mg2+, with depolarization. In contrast to Ca2+ influx through the plasma membrane, Ca2+ channels are not directly correlated with Vm when they are due exclusively to Ca2+ flux through internal membranes, such as those of the endoplasmic reticulum. In this case, Ca2+ flux does not contribute to the current underlying the membrane potential change. The second aspect addressed in this chapter concerns the calibration of the voltage-sensitive dye signal, in terms of a Vm change on an absolute scale (in mV). It must be said that in many instances, this calibration is not critical; the relevant information in many experiments can be derived from relative changes in optical signals recorded under different conditions. Indeed, commonly used fast potentiometric membrane dyes undergo a large charge shift upon excitation and display a strictly linear electrochromic response to membrane potential in the range of –100 mV to 100 mV (Loew and Simpson 1981). Therefore, at each individual site, the fractional change in fluorescence intensity under different conditions can be directly compared. The calibration of the voltage-sensitive dye signal becomes crucial, however, when the experiment requires the comparison of Vm signal amplitudes from different sites. In other words, an absolute calibration is necessary for the construction of spatial maps of the Vm signal amplitudes. The voltage-sensitive dye signals in this chapter, unless specified otherwise, are shown as the fractional change in fluorescence intensity

DF/F = (F(t) – F(t0))/F(t0), where F(t) is the recorded fluorescence intensity at a given time and F(t0) is the fluorescence intensity at the resting membrane potential (resting fluorescence). Although the calibration of voltage-sensitive dye signals as well as combining voltage and calcium imaging have general applicability, this chapter will pay particular attention to recordings from individual neurons, described in detail in the previous chapter.

4.2  Combining Voltage and Calcium Imaging 4.2.1  P  rinciples of Combined Imaging Using Two Indicators Combined fluorescence measurements are widely used in biology. In molecular biology, the concomitant localization of two or more proteins is achieved by using fluorescent antibodies with different absorption and emission spectra. Combined imaging of two different physiological parameters has been used less frequently. For instance, combined optical recordings using two indicators have been utilized to measure intracellular pH and Ca2+ (MartinezZaguilan et al. 1991) as well as slow membrane potential changes and Ca2+ signals (Kremer et al. 1992). Ideally, fluorescence signals from two indicators can be recorded independently in three different ways, depending on their optical properties. First, the two indicators may have separate absorption spectra but a largely overlapping region in the emission spectra. Second, the two indicators may have a large overlapping region in their absorption spectra but well separated emission spectra. Third, the two indicators may have well separated both the absorption and the emission spectra. Figure 4.1 shows the spectral representations for these three different cases and the optical arrangements to combine the two signals. In the first case (Fig. 4.1A), the fluorescence from the two dyes can be excited using two separate wavelengths, either from a single instrument (for example, a monochromator) or from two ­different light sources using a dichroic mirror. In either one of the two possible arrangements, the emitted light from the two indicators is recorded using a single detector and it is never possible to achieve a true simultaneous measurement. It is possible, however,

Marco Canepari  •  Division of Pharmacology and Neurobiology, Biozentrum-University of Basel, Klingelbergstrasse 50-70, CH-4056 Basel, Switzerland Peter Saggau  •  Department of Neuroscience, Baylor College of Medicine, Houston, TX 77030, USA Dejan Zecevic  •  Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, CT 06520, USA M. Canepari and D. Zecevic (eds.), Membrane Potential Imaging in the Nervous System: Methods and Applications, DOI 10.1007/978-1-4419-6558-5_4, © Springer Science+Business Media, LLC 2010

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Chapter 4    M. Canepari et al. ●

FIGURE 4.1.  Alternatives for combined imaging using two-indicators. (A) (Left) Ideal absorption and emission spectra of two indicators with separate absorption spectra and overlapping emission spectra. Arrows indicated by “ex” and “em” are positioned over ideal excitation and emission. Rectangles indicated by “d” are in position of ideal dichroic mirrors. (Right) Optical arrangements for excitation and recording of fluorescence using either one or two light sources. (B) (Left) Same as (A) but for two indicators with overlapping absorption spectra and separate emission spectra. (Right) Optical arrangements for excitation and recording of fluorescence using either one or two light detection systems. (C) (Left) Same as (A, B) but for two indicators with separate absorption and emission spectra. (Right) Optical arrangements for excitation and recording of fluorescence using either one or two light sources and light detection systems.

to alternate between the two different excitation wavelengths at relatively high speed. In the second case (Fig. 4.1B), because the excitation spectra are overlapping, fluorescence from both indicators can be excited using the same excitation wavelength. Emitted light can be recorded either by the same detector, by switching between two emission filters, or by two different detectors, after splitting the emitted light with a secondary dichroic mirror. In this second arrangement, by synchronizing data acquisition of the two detection systems, it is possible to achieve simultaneous imaging of the fluorescence signals from the two indicators. Finally, in the third case (Fig. 4.1C), the combined excitation and detection of the two fluorescence systems can be done either using one broad-band light source and one detection system or two light sources and two detection systems. In the first case, the excitation using the same light source requires changing the excitation filter and the dichroic mirror as well as changing the emission filter prior to detection. Thus, the two measurements cannot be done simultaneously. In the second arrangement, (two light sources and two detectors), the combined measurement of the two fluorescence signals can be, in principle, done simultaneously. The optimization of recording signals from two indicators depends on the availability of probes with narrow excitation and emission bands. This is not the case for the available fast voltage sensitive dyes. Most of the styryl voltage dyes are characterized by a broad excitation spectrum in the visible range and a broad emission spectrum in the red-IR region (Fluhler et al. 1985). Figure 4.2 shows the spectra of the hydrophobic di-8-ANEPPS which are similar to those of di-4-ANEPPS and of the water soluble indicators JPW-1114 (di-2-ANEPEQ) and JPW-3028 (all based on the same chromophore). Similar spectral properties are also shared by many of the commonly used “RH” dyes such as RH-795, RH-237, RH-421 and RH-414. With these dyes, well-separated spectra are

difficult to obtain using commercially available Ca2+ indicators excited in the visible spectral range. Nevertheless, the combined recordings are still possible due to a large Stokes shift of chargeshift voltage probes (~150 nm). In addition, more efficient combined recordings can be achieved using calcium indicators excited in the UV spectral range. Both voltage sensitive dyes and calcium indicators can be loaded into cells either by extracellular bath application of the indicator or by intracellular injection using either a patch-electrode or a sharp microelectrode. For extracellular loading, calcium dyes are commercially available in membrane-permeant AM-ester form. Once in the cell, the acetyl-ester is hydrolyzed by the endogenous esterases and the released free indicator remains in the cytoplasm (Yuste 2000). For intracellular loading, calcium indicators are available as water-soluble potassium salts. When injected into cells from a patch electrode, the dye can quickly equilibrate in the cytosol at a given concentration permitting quantitative estimate of Ca2+ signals (Eilers and Konnerth 2000). In general, intracellular loading of the calcium indicator is done in conjunction with the intracellular loading of the voltage sensitive dye. It is also possible to load the cell with the voltage sensitive dye first and include the calcium indicator in the pipette used for re-patching the labeled cell. In this protocol, voltage imaging is carried out first, immediately after re-patching, during which time the cell is loaded with the calcium indicator from the recording pipette.

4.2.2  C  ombining Voltage Dyes with Calcium Indicators Excitable in the Visible Range The overlapping excitation and separate emission spectra are obtained when using styryl voltage dyes in combination with green-emitting calcium indicators such as Fluo, Calcium Green and Oregon Green (see Fig. 4.2 for Calcium Green). The choice

Combined Voltage and Calcium Imaging and Signal Calibration     Chapter 4 ●

FIGURE 4.2.  Absorption and emission spectra of di-8 ANEPPS in combination with the ideal absorption and emission of Calcium Green and Fura calcium indicators. Left and right curves are the absorption and emission spectra as reported by Invitrogen – Molecular Probes. The thick downward arrow represents the excitation of the ratiometric indicator Fura. The thin downward arrows represent the excitation peak of Calcium Green. The upward arrows represent the emission peaks of the two indicators.

of the optimal calcium indicator depends on several factors. First, from the variety of commercially available dyes, it is possible to choose an indicator with optimal optical responsiveness and affinity for Ca2+ (this aspect is specifically addressed for UV-excitable indicators in Sect. 4.2.3). Second, it is possible to excite both voltage and calcium fluorescence using a single light source (for instance, a 488 nm laser; Bullen and Saggau 1998), permitting simultaneous voltage and calcium imaging. The sensitivity of voltage imaging is, however, sacrificed in this recording mode because 488 nm is not an optimal excitation wavelength for styryl voltage-sensitive dyes. Figure 4.3A shows the combined spectra of JPW-1114 and Calcium Green-1. By comparing high-affinity calcium indicators, Calcium Green-1 is preferable to Oregon Green BAPTA-1 because it has a lower overlap in the emission spectrum with JPW-1114 and to Fluo-3 because this indicator has low resting fluorescence and weak signal strength, which translates into a significant level of baseline noise (Bullen and Saggau 1998). Combined voltage and calcium imaging can also be done using laser-scanning microscopy (Bullen et al. 1997) with a 488 nm laser as illustrated by the diagram in Fig. 4.3B. This system is a particular implementation of the general configuration illustrated in Fig. 4.1B. An example of voltage and calcium fluorescence signals, from a cultured hippocampal pyramidal neuron, is shown in Fig. 4.3C. Combined voltage and calcium imaging from individual neurons using blue-excitable/green-emitting calcium indicators has also been used in several other studies. In the barrel cortex, the high-affinity indicator Oregon Green BAPTA-1 was combined with the voltage sensitive dye JPW-1114 in recordings from individual neurons and with the voltage indicator RH-1691 in in  vitro and in  vivo network recordings (Berger et  al. 2007). In prefrontal cortical neurons, dendritic recordings were obtained

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combining JPW-1114 either with the high-affinity calcium indicator Calcium Green-1 or with the low-affinity calcium indicator Fluo-5F (Milojkovic et al. 2007). With voltage-sensitive dyes having longer-wavelength excitation and emission spectra, simultaneous voltage and calcium imaging can been done using the calcium indicator Calcium Orange, which has excitation peak at ~550 nm and emission peak at ~580 nm (Sinha et al. 1995; Sinha and Saggau 1999). Combining voltage and calcium imaging using calcium indicators excited at the same wavelength as the voltage sensitive dye has, however, several disadvantages. First, a small but still significant overlap between the two emission spectra is still present (see Fig. 4.3A). This overlap does not preclude qualitative calcium imaging (as reported above) but may limit a quantification of calcium fluorescence signals (see Sect. 4.2.3). Another limitation related to the single excitation source is that the two dyes are usually characterized by significantly different brightness. Thus, the small “bleeding” of a strong voltage signal into a calcium recording channel can contaminate the weak calcium signal. In some situations, such emission cross talk can be mathematically corrected (Sinha et al. 1995). In general, this limitation determines a minimal concentration of the calcium indicator under which the calcium signal is significantly distorted by the voltage signal (Bullen and Saggau 1998). As mentioned before, although the concentration of the calcium indicator can be controlled in cells loaded by intracellular application of the probe, the final concentration of free indicator in cells loaded through AM-esters is limited and can be insufficient for this application. Finally, voltage and calcium signals have different time-course. While voltage recordings of synaptic or action potentials can be often achieved within 100–200 ms, recording of the complete time-course of the corresponding calcium signal requires longer acquisition intervals (Canepari et al. 2008). Thus, the voltage sensitive dye must be exposed to light for a much longer time than what would be required for the voltage measurement alone, increasing both bleaching of the dye and photo-toxicity. The above-mentioned limitations can be overcome by using a calcium indicator excited at a wavelength not absorbed by the voltage-sensitive dye, as described in Sect. 4.2.3.

4.2.3  C  ombining Voltage Dyes with Calcium Indicators Excitable by UV Light For styryl voltage sensitive dyes such as di-4-ANEPPS, di-8ANEPPS and Di-2-ANEPEQ (JPW-1114), the minimal overlapping in the absorption and emission spectra is obtained using calcium indicators excitable in the UV range (Fig. 4.2). The use of one of these indicators minimizes the contamination of the calcium indicator fluorescence by the fluorescence from the voltage-sensitive dye. Additionally, the voltage-sensitive dye is not excited during Ca2+-imaging, preventing a substantial photodynamic damage that can occur during relatively long recording periods (Canepari et al. 2008). UV-excitable Fura indicators with different equilibrium constants (Kd) are commercially available (see Table 4.1). This permits the choice of the most suitable indicator for the measurement of either the Ca2+ influx (high-affinity indicator) or the change in intracellular free Ca2+ concentration (low-affinity indicator). In contrast to the indicators excited in the visible range, Fura indicators allow ratiometric measurements. When excited above their isosbestic wavelength (~360 nm), the fractional changes of fluorescence (DF/F) corresponding to an increase in Ca2+ concentration are negative. Imaging above the isosbestic wavelength has several advantages. First, the resting fluorescence, which corresponds to nominally

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Chapter 4    M. Canepari et al. ●

FIGURE 4.3.  Combining voltage and calcium imaging using JPW-1114 and Calcium Green-1. (A) Combined excitation and emission spectra of JPW-1114 (dotted line) and Calcium Green-1 (straight line) with the relative placement the emission filters em1 and em2. (B) Epifluorescence configuration for high-speed randomaccess, laser-scanning microscopy (similar to the configuration reported in Fig. 4.1b). (C) Representative voltage and calcium signals simultaneously recorded from the proximal dendrite of a cultured hippocampal pyramidal neuron; action potentials were elicited by current injection through a somatic patch pipette also used for somatic recording. Reproduced from Bullen and Saggau (1998) with the permission of Springer.

TABLE 4.1.  Dissociation Constants and Buffering Capacities of Fura Dyes Dye

Kd (mM)

Kdye (300 mM)

Kdye (1 mM)

fura-2 fura-5F bis-fura-2 fura-4F fura-6F fura-FF Mag-fura-2

0.224a 0.4a 0.525a 0.77a 5.3a 10b 25c–40d

~1,300 ~750 ~570 ~390 ~57 ~30 ~7.5–12

~4,500 ~2,500 ~1,900 ~1,300 ~190 ~100 ~25–40

A list of commercially available Fura dyes with dissociation constant (Kd) and buffering capacities (Kdye) at the two concentrations of 300 µM and 1 mM. a  Invitrogen–Molecular Probes handbook (for fura-2 and bis-fura-2 at 1 mM Mg2+) b  Schneggenburger et al. (1999) c  Hyrc et al. (2000) d  Naraghi (1997)

0 Ca2+, is significantly higher than with dyes that increase their fluorescence with Ca2+. Second, the achievable imaging contrast is high because healthy cells are characterized by very low resting Ca2+, and the dye in adjacent structures, exposed to millimolar Ca2+ concentrations, is essentially not fluorescent. Third, since the dynamic range of the fluorescence is ~1 after subtraction of fluorescence at saturating Ca2+, these measurements allow a better quantitative estimate of the

Ca2+ signals. An example of voltage and Ca2+ optical signals obtained sequentially from the two indicators from three regions on the dendritic tree of a Purkinje neuron stained by JPW-1114 and Fura-FF is illustrated in Fig. 4.4. Using an excitation band of 387 ± 5 nm for Fura-FF (Fig. 4.4A), the Ca2+ increase following the stimulation of the climbing fibre corresponds to a fractional decrease of fluorescence (Fig. 4.4B). Although simultaneous voltage and calcium imaging using UV-excitable calcium indicators is possible in principle (see Fig. 4.1C), up to now the combined recording has been done only by sequential imaging in CA1 hippocampal pyramidal neurons (Canepari et  al. 2007), in prefrontal cortex layer-5 pyramidal neurons (Milojkovic et al. 2007) and in cerebellar Purkinje neurons (Canepari and Vogt 2008). Sequential recording (as well as signal averaging) is meaningful only if repeated application of the same stimulation protocol results in the same response. This requirement must be confirmed experimentally by comparing individual recor­ dings as shown in measurements of Fig. 4.4C. In this example, the voltage and the Ca2+ optical signals were recorded from a dendritic location on a cerebellar Purkinje neuron in response to four repetitions of climbing fibre activation separated by 1 min. The results showed that signals were practically identical in four individual trials (gray traces), permitting the correlation of voltage and Ca2+ optical signals recorded sequentially in response to the same stimulus.

Combined Voltage and Calcium Imaging and Signal Calibration     Chapter 4 ●

FIGURE 4.4.  Combining voltage and calcium imaging using JPW-1114 and a  UV excitable calcium indicator. (A) Schematic of the imaging apparatus for  sequential voltage and calcium imaging using a Fura indicator. Filters for ­voltage-sensitive dye (JPW): ex1 = 525 ± 25 nm, d1 > 570 nm and em1 > 610 nm. Filters for Ca2+ indicator (Fura): ex2 = 387 ± 6 nm, d1 > 470 nm and em1 = 510 ± 42 nm. (B) Vm and Ca2+ fractional changes of fluorescence from cerebellar Purkinje neuron dendrites related to a climbing fibre EPSP recorded from the locations 1–3 reported in the fluorescence images on the left. Somatic recording of the climbing fibre EPSP is the upper trace. (C) Individual Vm (left) and Ca2+ (right) fractional changes of fluorescence (gray traces) related to a climbing fibre EPSP. Superimposed black traces are the averages of the four trials. Reproduced from Canepari, Vogt and Zecevic (2008) with the permission of Springer.

An important aspect of combined voltage and calcium imaging is the interpretation of Ca2+ optical signals, which depends on how much the Ca2+ indicator perturbs the physiological Ca2+ homeostasis. The buffering capacity of a Ca2+ indicator, Kdye, defined as the ratio between the dye-bound Ca2+ and the free Ca2+ in the presence of the indicator depends on the dissociation constant and on the concentration of the indicator (Table 4.1). The perturbation of the physiological Ca2+ introduced by the Ca2+ indicator can be evaluated by comparing the parameter Kdye with the endogenous buffering capacity of the cell (Kcell). The interpretation of Ca2+ optical signals is

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simplified when Kdye is either much larger or much smaller than Kcell, i.e. when most of Ca2+ binds to the indicator or when the fraction of Ca2+ bound to the indicator is negligible compared to the total Ca2+ (Canepari et al. 2008). Without addressing in detail the issue of calibration of calcium optical signals (Neher 2000) in this volume focused on Vm imaging, we will note that, in the first case, the fractional change of Ca2+ related fluorescence (DF/F) is approximately linear with the total intracellular Ca2+ signal. In the second case, the time course of the ratio (F–Fmin)/(Fmax–F), where Fmin and Fmax are the fluorescence intensities at 0 and saturating Ca2+ respectively, is linear with the physiological intracellular free Ca2+ concentration change (D[Ca2+]i). In many instances, Ca2+ signals are due to Ca2+ influx through a Ca2+ channel in the plasma membrane. The contribution to the change in membrane potential due to the Ca2+ influx depends on the Ca2+ permeability of the channel relative to its permeability to other ions, in particular to Na+ and K+. This is different for VGCCs, AMPA receptors, NMDA receptors and other Ca2+ permeable pores such as transient receptor potential (TRP) channels. Vice-versa, because opening or unblocking of Ca2+ channels often depends, in a non-linear manner, on the local membrane potential, the Ca2+ influx is often, but not always, larger when the voltage related depolarizing optical signal is larger (Canepari et al. 2007). Because this bi-directional relationship is complex, the analysis of voltage and calcium signals from multiple sites on individual neurons is not straight-forward and requires careful interpretation. In synaptic plasticity studies, a quantity of considerable interest is the supra-linear Ca2+ signal. The supra-linear Ca2+ signal occurs when two stimulation protocols are combined to evoke the coincident activity (pairing protocol) which results in a Ca2+ signal that is larger than the sum of the two Ca2+ signals associated with the application of individual stimulation protocols. A supra-linear Ca2+ signal is always caused by a supra-linear D[Ca2+]i, but not necessarily by a supra-linear Ca2+ influx through the plasma membrane. Indeed, the supra-linear Ca2+ signal might be caused by Ca2+ release from internal stores or by the saturation of the endogenous Ca2+ buffer. However, in the latter case, if the buffering capacity of the dye dominates over the endogenous buffering capacity of the cell and the dye is not saturated, the presence of the indicator will cancel the supra-linear D[Ca2+]i and no Ca2+ dependent DF/F will be observed. Thus, in these conditions, detection of supra-linear Ca2+ dependent DF/F signals which are not due to Ca2+ release from internal stores always corresponds to a supra-linear Ca2+ influx and it must always correlate with an increase in the depolarizing voltage signal. On the contrary, if the buffering of the dye is negligible compared to the endogenous buffering of the cell, a supra-linear D[Ca2+]i signal that is due to the saturation of the endogenous Ca2+ buffer can be measured reliably. The two different types of supra-linear Ca2+ signals not involving Ca2+ release from stores are illustrated by the two following examples. In measurements from CA1 hippocampal pyramidal neurons (Canepari et al. 2007), cells were loaded with the voltage-sensitive dye JPW-3028 and 300 mM Bis-Fura-2 to analyze, over large regions of the dendritic tree, the voltage and Ca2+ optical signals associated with back-propagating action potentials, with excitatory postsynaptic potentials (EPSPs) and with pairing of these two membrane potential transients using an LTP induction protocol. Pyramidal neurons in the hippocampal CA1 region are characterized by relatively low endogenous buffering capacity in the dendrites (~100) and even lower buffering capacity (~20) in the spines (Sabatini et al. 2002). Thus, the buffering capacity of 300 mM BisFura-2 (Kd ~ 500 in the presence of Mg2+, see Table 4.1) is ~6 times larger than the buffering capacity of the dendrite and ~30 times

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larger than the buffering capacity of the spine. Ca2+ signals associated either with back-propagating action potentials or with EPSPs were mediated by Ca2+ influx through voltage-gated calcium channels and/or through NMDA receptors. As shown in Fig. 4.5A, the pairing of the two stimulating protocols elicited a supra-linear Ca2+ signal. In the presence of the NMDA receptor blocker AP-5, since the measurements were done using a non-saturating concentration of a high-affinity indicator, the supra-linear Ca2+ signal must have been caused by supra-linear Ca2+ influx mediated by recruitment of additional voltage-gated Ca2+ channels. In agreement with this expectation, at each site where a supra-linear Ca2+ signal was observed, the Vm related optical signal during the pairing protocol had a larger peak depolarization compared to the signals associated with unpaired stimulations (Fig. 4.5A). In measurements from cerebellar Purkinje neurons (Canepari and Vogt 2008), cells were loaded with 1 mM of the Ca2+ indicator Fura-FF and with the voltage sensitive dye JPW-1114. Purkinje neurons have an exceptionally high dendritic Kcell estimated at ~2,000 (Fierro and Llano 1996). Therefore, the addition of a low affinity Ca2+ indicator such as Fura-FF (Kd ~ 10), even at millimolar concentrations, does not significantly alter the physiological homeostasis and it is possible to record changes in free intracellular calcium concentration (D[Ca2+]i signals (Canepari et al. 2008)). In this particular preparation, Vm signals were calibrated in terms of membrane potential as described in Sect. 4.3.2. As shown in Fig. 4.5B, pairing the stimulation of the large climbing fibre synaptic potential with local

parallel fibres stimulation generated a local dendritic supra-linear D[Ca2+]i signal which was independent of Ca2+ release from stores (Brenowitz and Regehr 2005). Although the D[Ca2+]i signal originated from Ca2+ influx through voltage-gated calcium channels, in Purkinje cells, in contrast to CA1 pyramidal neurons, the Vm signal during the pairing protocol did not have larger peak depolarization compared to the Vm signals associated with unpaired stimulations (Fig. 4.5B). In this case the Ca2+ influx associated with the climbing fibre synaptic potential was the same in paired and unpaired conditions. The corresponding D[Ca2+]i signal, however, was larger following paired stimulation because the Ca2+ influx associated with the parallel fibre stimulation locally and transiently saturated the endogenous Ca2+ buffer (Canepari and Vogt 2008). This supra-linear Ca2+ signal could be correctly interpreted only when recorded using a low-affinity indicator. When the cell was injected with 10 mM Bis-Fura-2, Kdye became ~10 times larger than Kcell and supra-linear Ca2+ signals were abolished (Canepari and Vogt 2008).

4.2.3.1  O  ther Possibilities for Combining Voltage and Calcium Imaging

In the previous paragraphs we have described fluorescence voltage sensitive dyes excited by a collection of wavelengths in the range of 475–565 nm and emitting in the range of 600–700 nm. The use of voltage-sensitive dyes that are excited at longer wavelengths can expand the possibilities in combining voltage imaging with

FIGURE 4.5.  Combining voltage and calcium imaging using high- and low-affinity Fura indicators. (A) (Left) Ca2+ and Vm fractional changes of fluorescence corresponding to back-propagating action potentials (bAPs) and paired EPSP–bAP signals from two locations on the dendritic tree of a CA1 hippocampal pyramidal neuron. Signals are superimposed to reveal the region-specific increase in peak depolarization during paired activity. (Right) Fluorescence image of the dendritic arbor of three hippocampal pyramidal neurons. The stimulating electrode is indicated schematically. (Middle and bottom panels) Color-coded spatial maps of the Ca2+ signal and of the Vm signal during paired activity respectively; signals were scaled using the signals in unpaired conditions. Experiments are in the presence of the NMDA receptor blocker AP-5. Supra-linear Ca2+ correlated with larger depolarization. Reproduced from Canepari et al. (2007) with the permission of Wiley-Blackwell. (B) (Top) Recorded dendrites of a cerebellar Purkinje neuron; supra-linear D[Ca2+]i from the difference between D[Ca2+]i associated with the pairing protocol and D[Ca2+]i associated with the unpaired PF-EPSPs and CF-EPSP in color-coded scale. (Bottom) D[Ca2+]i and DVm from the square region depicted above associated with 1 unpaired (black traces) and 1 paired CF-EPSP. Supra-linear Ca2+ not corresponding to larger depolarization. Modified from Canepari and Vogt (2008).

Combined Voltage and Calcium Imaging and Signal Calibration     Chapter 4 ●

calcium imaging. New longer-wavelength water-soluble dyes for selective staining of individual neurons by intracellular injection have been recently introduced (Zhou et al. 2007). These molecules, excitable at 600–700 nm, have a smaller spectral overlap with calcium indicators excitable by blue light, thus allowing for better selection of indicators in combined imaging. Another possibility is the combination of an absorption voltage sensitive dye with a fluorescent calcium indicator. Using bath application of the absorption voltage sensitive dye RH-482 and a low-affinity calcium indicator Magnesium Green, Sabatini and Regehr could correlate the Ca2+ signal underlying synaptic release with the excitation of pre-synaptic terminals in cerebellar synapses (Sabatini and Regehr 1996, 1997). In these experiments, simultaneous calcium and voltage measurements were done by using excitation light at ~500 nm and recording fluorescence from the calcium indicator Magnesium Green and the transmitted light from voltage-sensitive dye RH-482 at 710 nm with two photodiodes (no spatial resolution). The important limitation of this approach is that the absorption measurements from multiple sites on individual neurons are substantially less sensitive compared to fluorescence measurements (Antic and Zecevic 1995). In the above example, the reported successful recordings of absorption AP signals from presynaptic terminals were possible because the nature of the experiment allowed extensive temporal and spatial signal averaging over a large region of the cerebellar slice (Sabatini and Regehr 1997).

4.3  Calibration of Voltage Optical Signals The calibration of Vm optical signals on an absolute scale (in mV) provides direct measurements of Vm signal amplitudes from multiple locations. This information can be used to construct and analyse the dynamic spatial maps of signal amplitudes throughout neuronal processes. The temporaly and spatially well resolved maps of activity are one of the key elements in understanding the rules governing dendritic signal processing and plasticity. The absolute calibration of optical Vm signals, however, is not straight-forward and often the exact callibration is not possible. Voltage-sensitive dye recording of membrane potential transients belongs to a class of indirect measurements. The quantity that is being measured directly, by photodetectors, is light intensity, and the quantity that needs to be monitored (membrane potential) is derived from a known relationship between the light intensity and the membrane potential. It is convenient that the relationship between light intensity and membrane potential is strictly linear over the entire physiological range for many voltage sensitive dyes (e.g. Gupta et al. 1981; Loew and Simpson 1981; Wu and Cohen 1993). For that reason, for measurements from one individual site, if an electrical measurement is done simultaneously with the optical measurement (as in squid giant axon experiments) the absolute calibration is automatically obtained. Then, the electrode could be removed and all further optical recordings would be precisely calibrated in terms of voltage. A more complicated situation arises when optical measurements of Vm transients are done from multiple (e.g. several hundreds) sites on the object. Because the number of sites accessible with electrodes is practically limited, the simple calibration procedure described above is not possible. In a multisite recording, the fractional change in light intensity is still proportional to voltage, but also, to a different extent at different sites, to an additional factor: the ratio of inactive dye to active dye. The inactive dye is bound to connective tissue or any other membranes that do not change potential while the active dye is bound to the excitable

49

membrane being monitored. The inactive dye contributes to the resting fluorescence only, and the light from active dye contributes to the resting fluorescence and also carries the signal. Obviously, if all the dye is in the excitable membrane, the optical signal expressed as the fractional fluorescence change (DF/F) will be 10 times bigger, for the same change in membrane potential, than if there is 10 times more inactive dye than active dye. This is an essential consideration that explains why extracellular dye application is dramatically inferior, in terms of S/N ratio, to intracellular staining of individual nerve cells. Extracellular staining generates a large excess of inactive dye that results in large background fluorescence and reduced DF/F signal. If the voltage-sensitive dye is bound exclusively to excitable membranes the differences in fluorescence intensity change (DF) recorded from different sites in response to a constant voltage transient are caused solely by the differences in the intensity of resting fluorescence (F) projected to different detectors (due to differences in the amount of dye in different regions). This factor is eliminated by normalizing the signals to the resting light intensity for each individual pixel; dividing the signal (DF) by resting light intensity (F) for each detector will equalize the sensitivity of all elements in the photodetector array. In that case, calibration of the optical signal in terms of membrane potential from any one location (which is usually easy) by simultaneous optical and electrical recording will be valid for the whole array. One deviation from this rule arises in situations where autofluorescence (not related to voltage-sensitive dye) from the object is not negligible and contributes significantly and to a diffe­ rent degree to the resting fluorescence recorded by each detector. The special case in which all of the dye responds to Vm change is rarely found. Generally, both active and inactive dye contribute to fluorescence light. For example, in experiments utilizing intracellular application of the dye, inactive dye would be bound to intracellular membranes and organelles. Furthermore, it is a general rule that the ratio of active dye to inactive dye is different for different regions of the object and is also unknown. It follows that the sensitivity of recording from different regions of the object will be different and the calibration of all detectors cannot be done by calibrating the optical signal from any single site. In this situation, the amplitude calibration of optical signals in terms of voltage will require separate calibration of each pixel to determine the sensiti­ vity profile of the array (see below).

4.3.1  R  atiometric Calibration of Voltage-Sensitive Dyes Signals It has been demonstrated that the voltage across membranes in different preparations is linearly related to the ratio of fluorescence excited from the two wings of the absorption spectrum of a voltage-sensitive dye which responds via a rapid spectral shift to membrane potential changes (Montana et  al. 1989). Thus, it is possible to use dual-wavelength ratiometric methods in Vm-imaging in a way analogous to ratiometric measurements developed for cation indicators (Grynkiewicz et al. 1985). While the resultant change in fluorescence intensity monitored at a single wavelength and expressed as a fractional change (DF/F) is useful for measurements of voltage transients such as action potentials, the dual-wavelength ratiometric measurement extends the usefulness of fast potentiometric dyes by filtering out complex or artificial changes in fluorescence intensity and providing a voltage-dependent signal that is internally standardized. In this way, the ratiometric approach can provide a direct measure of the resting membrane potential which is not restricted to measuring Vm changes. However, it is important to emphasize that calibration must be carried out for each specific

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application and for each recording location because different staining and background levels, filters, and light sources will provide different light intensities at the two excitation wavelengths. According to the property of the indicator, ratiometric calibration can be done either by measuring the ratio of the fluorescence emission at two different excitation wavelengths, or by measuring two emission wavelengths at a given excitation wavelength. The first approach can be done using one of the two optical configurations shown in Fig. 4.1A. The second approach, requiring one of the configurations shown in Fig. 4.1B, allows simultaneous recordings of the two emission wavelengths if two detectors are used. The excitation spectral shift has been used as the basis for ratiometric determinations of the resting membrane potential in various cell types and preparations (Bedlack et al. 1992; Gross et al. 1994; Zhang et al. 1998). Using slow dual wavelength excitation, it was possible to calibrate the membrane potential in neuroblastoma N1E-115 cells (Zhang et  al. 1998). In that case, the cells were stained extracellularly with di-8-ANEPPS and fluorescence excited either by 440 ± 15 nm or 530 ± 15 nm light. Because the relationship between the ratio of signals at two wavelengths (R) and the absolute value of the membrane potential was consistent for all the cells of this type (Zhang et  al. 1998), ratiometric measurement allowed for the direct conversion of the voltage fluorescence into the corresponding absolute value of the membrane potential. Ratiometric Vm imaging at two excitation wavelengths is limited in temporal resolution by the time required to process interlaced images or to switch between the two excitation lines. These limitations do not apply to voltage ratiometric imaging using two emission wavelengths. The shift in the emission spectrum of di-8-ANEPPS, accurately measured in the endothelium of intact arterioles (Beach et al. 1996) and in human embryonic kindney HEK 293 cells (Kao et al. 2001) by processing the emitted light from the preparation with a prism spectrograph, allows for dual emission ratiometric imaging. An example of optical arrangement to perform dual-emission ratiometric imaging calibrated in terms of membrane potential on an absolute scale is illustrated in the example of Fig. 4.6 (Bullen and Saggau 1999). In this example, the excitation of neurons stained extracellularly with di-8-ANEPPS was provided by a laser, either at 476, 488 or 502 nm, and the emission was separated by the secondary dichroic mirror to obtain the fluorescence ratio at 520 ± 25 nm and 620 ± 30 nm. Fluorescence measurements are often contaminated by the noise from the light source or by slow bleaching artifacts (commonmode noise), as well as by slow movements of the preparation. A further advantage of ratiometric imaging using simultaneous recordings of two emission wavelengths is the cancellation of dye bleaching and common-mode noise (Bullen and Saggau 1999). In addition, dual emission ratiometric voltage imaging considerably improves measurements of changes of membrane potential in preparations where motion artifacts are non-negligible such as the arterioles (Beach et al. 1996) or the heart (Knisley et al. 2000).

4.3.2  C  alibration of Voltage-Sensitive Dye Signals by Using Reference Signal of Known Amplitude In multisite measurements where a significant fraction of fluorescence originates from inactive dye and from non-uniform background fluorescence, simple calibration of optical signals in terms of Vm is not possible. However, the calibration is absolute and straightforward if a calibrating electrical signal that has known amplitude at all locations is available. An all-or-none action

FIGURE 4.6.  Schematic representation of an optical arrangement to produce ratiometric recordings at two emission wavelengths. Fluorescence records (DF/F) for each spectral band are shown in the left and right boxes. Ratio formation and filtering were completed digitally before the records were displayed interactively as DR/R. The final normalized and calibrated trace is shown in the bottom box. Laser lines used included 476 nm, 488 nm, and 502 nm. The secondary dichroic mirror (d2) separated light emission 570 nm. The two light beams were filtered at 540 ± 25 nm (em1) and at 600 ± 30 nm (em2). Reproduced from Bullen and Saggau (1999) with the permission of Elsevier.

potential signal is ideal for this purpose and can be used to create a sensitivity profile of the measuring system. This type of calibration was used to scale the amplitudes of sub-threshold signals in mitral cells of the olfactory bulb. In this system, the action potential is constant in amplitude along the entire length of the primary dendrite (Bischofberger and Jonas 1997; Chen et al. 1997). This calibration protocol is illustrated in Fig. 4.7A and B. In this experiment, the amplitude of the spike signal was determined from a patchelectrode recording from the soma and correlated to the optical signal at each individual site. Using this information, it was possible to measure the amplitude of sub-threshold synaptic potentials at different dendritic sites (Djurisic et al. 2004) and to derive the functional structure of the dendritic tuft (Djurisic et al. 2008). If action potentials are not available, another type of calibrating electrical signal that has known amplitude at all locations is necessary. One idea is to take advantage of the fact that slow electrical signals spread with minimal attenuation over distances that can be relatively long in some neuronal classes. Such condition occurs, for instance, in cerebellar Purkinje neurons (Roth and Häusser 2001). Calibration of optical signals using prolonged hyperpolarizing pulses was utilized to measure local dendritic climbing fiber and parallel fiber synaptic potentials (Canepari and Vogt 2008). This calibration procedure is illustrated in Fig. 4.7C, D. Panel (C) shows

Combined Voltage and Calcium Imaging and Signal Calibration     Chapter 4 ●

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FIGURE 4.7.  Examples of calibrations of voltage-­ sensitive dye signals. (A) Image of a mitral cell. (B) (Left) Single-trial optical recordings from ten different regions on the primary and oblique dendrites showing signals corresponding to an evoked action potential. (Center) Calibration of optical signals (DF/F) in terms of membrane potential (in millivolts); top trace shows an optical signal corresponding to an action potential of 93 mV used as a calibration standard; middle trace is a subthreshold EPSP signal evoked by olfactory nerve stimulation; bottom trace is a threshold EPSP signal recorded from the tuft after the action potential was blocked by intracellular application of QX-314. (Right) The amplitude of EPSP signals on a voltage scale at ten recording sites calibrated using the sensitivity profile deter­ mined from action potential measurements. (C) (Left) Fluorescent image of the recorded dendrites with three sample regions (8 × 8 pixels). (Right) Fractional changes of voltage fluorescence from the three sample regions following a 10 mV hyperpolarizing pulse; somatic recording indicated. (D) Fractional changes of fluorescence (left) and calibrated DVm signals (right) in the three sample regions associated with a CF-EPSP; somatic recording indicated.

the fractional change of fluorescence corresponding to ~10 mV steady-state hyperpolarization evoked in three regions of the dendritic tree. Panel (f) shows the conversion from DF/F signals, associated with a climbing fiber synaptic potential into the local membrane potential changes. In other neurons with long and thin apical and basal dendrites, a hyperpolarizing signal will attenuate with distance significantly and cannot be used for calibration. Nevertheless, an approximate calibration of optical signals is sometimes still possible at many dendritic locations based on the absolute amplitude of the backpropagating action potential in different parts of the dendritic tree as determined in numerous dendritic patch-electrode recordings (e. g. Frick et al. 2004). Finally, Vm changes of known amplitude can be obtained by setting the resting membrane potential to the reversal potential of K+ and by changing the extracellular K+ concentration. This can be accomplished by increasing the membrane permeability to K+ using ionophores such as valinomycin. Using this approach, voltage ratiometric optical signals were converted to mV in neuroblastoma N1E-115 cells (Bedlack et al. 1992).

References Antic S, Zecevic D (1995) Optical signals from neurons with internally applied voltage-sensitive dyes. J Neurosci 15:1392–1405. Beach JM, McGahren ED, Xia J, Duling BR (1996) Ratiometric measurement of endothelial depolarization in arterioles with a potential-sensitive dye. Am J Physiol 270:2216–2227.

Bedlack RS, Wei M-D, Loew LM (1992) Localized membrane depolarizations and localized calcium influx during electric field-guided neurite growth. Neuron 9:393–403. Berger T, Borgdorff A et al. (2007) Combined voltage and calcium epifluorescence imaging in vitro and in vivo reveals subthreshold and suprathreshold dynamics of mouse barrel cortex. J Neurophysiol 97:3751–3762. Bischofberger J, Jonas P (1997) Action potential propagation into the presynaptic dendrites of rat mitral cells. J Physiol (Lond) 504:359–365. Brenowitz SD, Regehr WG (2005) Associative short-term synaptic plasticity mediated by endocannabinoids. Neuron 45:419–431. Bullen A, Saggau P (1998) Indicators and optical configuration for simultaneous high-resolution recording of membrane potential and intracellular calcium using laser scanning microscopy. Pflügers Arch 436:788–796. Bullen A, Saggau P (1999) High-speed, random-access fluorescence microscopy: II. Fast quantitative measurements with voltage-sensitive dyes. Biophys J 76:2272–2287. Bullen A, Patel SS, Saggau P (1997) High-speed, random-access fluorescence microscopy: I. High-resolution optical recording with voltage-sensitive dyes and ion indicators. Biophys J 73:477–491. Canepari M, Vogt KE (2008) Dendritic spike saturation of endogenous calcium buffer and induction of postsynaptic cerebellar LTP. PLoS ONE 3:e4011. Canepari M, Djurisic M, Zecevic D (2007) Dendritic signals from rat hip­ pocampal CA1 pyramidal neurons during coincident pre- and post-synaptic activity: a combined voltage- and calcium imaging study. J Physiol 580: 463–484. Canepari M, Vogt K, Zecevic D (2008) Combining voltage and calcium imaging from neuronal dendrites. Cell Mol Neurobiol 58:1079–1093. Chen WR, Midtgaard J, Shepherd GM (1997) Forward and backward propagation of dendritic impulses and their synaptic control in mitral cells. Science 278:463–467.

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Djurisic M, Antic S, Chen WR, Zecevic D (2004) Voltage imaging from dendrites of mitral cells: EPSP attenuation and spike trigger zones. J Neurosci 24:6703–6714. Djurisic M, Popovic M, Carnevale N, Zecevic D (2008) Functional structure of the mitral cell dendritic tuft in the rat olfactory bulb. J Neurosci 28: 4057–4068. Eilers J, Konnerth A (2000) Dye loading with patch pipets. In: Yuste R, Lanni F, Konnerth A (eds) Imaging neurons a laboratory manual. Cold Spring Harbour Laboratory Press, New York. Fierro L, Llano I (1996) High endogenous calcium buffering in Purkinje cells from rat cerebellar slices. J Physiol 496:617–625. Fluhler E, Burnham VG, Loew LM (1985) Spectra, membrane binding, and potentiometric responses of new charge shift probes. Biochemistry 24:5749–5755. Frick A, Magee J, Johnston D (2004). LTP is accompanied by an enhanced local excitability of pyramidal neuron dendrites. Nat Neurosci 7:126–135. Gross E, Bedlack RS, Loew LM (1994) Dual-wavelength ratiometric fluorescence measurement of the membrane dipole potential. Biophys J 67:208–216. Grynkiewicz G, Poenie M, Tsien RY (1985) A new generation of Ca2+ indicators with greatly improved fluorescence properties. J Biol Chem 260:3440–3450. Gupta RK, Salzberg BM et  al. (1981) Improvements in optical methods for ­measuring rapid changes in membrane potential. J Membr Biol 58: 123–137. Hyrc KL, Bownik JM, Goldberg MP (2000). Ionic selectivity of low-affinity ratiometric calcium indicators: mag-Fura-2, Fura-2FF and BTC. Cell Calcium 27:75–86. Kao WY, Davis CE, Kim YI, Beach JM (2001) Fluorescence emission spectral shift measurements of membrane potential in single cells. Biophys J 81: 1163–1170. Knisley SB, Justice RK, Kong W, Johnson PL (2000) Ratiometry of transmembrane voltage-sensitive fluorescent dye emission in hearts. Am J Physiol Heart Circ Physiol 279:1421–1433. Kremer SG, Zeng W, Skorecki KL (1992) Simultaneous fluorescence measurement of calcium and membrane potential responses to endothelin. Am J Physiol 263:1302–1309. Loew LM, Simpson LL (1981) Charge-shift probes of membrane potential: a probable electrochromic mechanism for p-aminostyrylpyridinium probes on a hemispherical lipid bilayer. Biophys J 34:353–365. Martinez-Zaguilan R, Martinez GM, Lattanzio F, Gillies R J (1991) Simultaneous measurement of intracellular pH and Ca2+ using the fluorescence of SNARF-1 and fura-2. Am J Physiol 260:297–307.

Milojkovic BA, Zhou WL, Antic SD (2007) Voltage and calcium transients in basal dendrites of the rat prefrontal cortex. J Physiol 585:447–468. Montana V, Farkas DL, Loew LM (1989) Dual-wavelength ratiometric fluorescence measurements of membrane potential. Biochemistry 28: 4536–4539. Naraghi M (1997) T-jump study of calcium binding kinetics of calcium chelators. Cell Calcium 22:255–268. Neher E (2000) Some quantitative aspects of calcium fluorimetry. In: Yuste R, Lanni F, Konnerth A (eds) Imaging neurons: a laboratory manual. Cold Spring Harbour Laboratory Press, New York. Roth A, Häusser M (2001) Compartmental models of rat cerebellar Purkinje cells based on simultaneous somatic and dendritic patch-clamp recordings. J Physiol 535:445–472. Sabatini BL, Regehr WG (1996) Timing of neurotransmission at fast synapses in the mammalian brain. Nature 384:170–172. Sabatini BL, Regehr WG (1997) Control of neurotransmitter release by presynaptic waveform at the granule cell to Purkinje cell synapse. J Neurosci 17:3425–3435. Sabatini BS, Oertner TG, Svoboda K (2002) The life cycle of Ca2+ ions in dendritic spines. Neuron 33:439–452. Schneggenburger R, Meyer AC, Neher E (1999) Released fraction and total size of a pool of immediately available transmitter quanta at a calyx synapse. Neuron 23:399–409. Sinha SR, Saggau P (1999) Simultaneous optical recording of membrane potential and intracellular calcium from brain slices. Methods 18:204–214. Sinha SR, Patel SS, Saggau P (1995) Simultaneous optical recording of evoked and spontaneous transients of membrane potential and intracellular calcium concentration with high spatio-temporal resolution. J Neurosci Methods 60:49–46. Wu JY, Cohen LB (1993) Fast multisite optical measurement of membrane potential. In: Mason WT (ed) Biological techniques: fluorescent and luminescent probes for biological activity. Academic Press, New York. Yuste R (2000) Loading brain slices with AM esters of calcium indicators. In: Yuste R, Lanni F, Konnerth A (eds) Imaging neurons: a laboratory manual. Cold Spring Harbour Laboratory Press, New York. Zhang J, Davidson RM, Wei M-D, Loew LM (1998) Membrane electric properties by combined patch clamp and fluorescence ratio imaging in single neurons. Biophys J 74:48–53. Zhou W-L, Yan P, Wuskell JP, Loew LM, Antic SD (2007) Intracellular longwavelength voltage-sensitive dyes for studying the dynamics of action potentials in axons and thin dendrites. J Neurosci Methods 164: 225–239.

5

Use of Fast-Responding Voltage-Sensitive Dyes for Large-Scale Recording of Neuronal Spiking Activity with Single-Cell Resolution William N. Frost, Jean Wang, Christopher J. Brandon, Caroline Moore-Kochlacs, Terrence J. Sejnowski, and Evan S. Hill 5.1  Introduction Efforts to understand how the brain works require tools for observing circuits in action. Conventional electrophysiological methods can monitor no more than a few neurons at a time, providing a severely restricted perspective on network function. Optical recording with fast voltage-sensitive dyes (fVSDs) offers a way to overcome this limitation, by revealing the action potentials of dozens to hundreds of individual neurons during behaviorally relevant motor programs (Fig. 5.1). The availability of methods for monitoring large-scale brain activity with single-cell and sub-millisecond resolution is likely to advance our understanding of network function far beyond that attained using traditional methods (Yuste 2008). It has been three decades since fVSDs were first used to monitor the simultaneous firing of multiple individual neurons (Grinvald et al. 1977; Salzberg et al. 1977). Although most such studies have used invertebrate preparations, particularly those with large neurons whose action potentials fully invade the soma (Boyle et  al. 1983; London et al. 1987; Cohen et al. 1989; Zecevic et al. 1989; Nakashima et  al. 1992; Nikitin and Balaban 2000; Zochowski et  al. 2000a; Brown et al. 2001; Kojima et al. 2001; Frost et al. 2007; Wu et al. 1994a), a few studies have accomplished this in vertebrate preparations, such as the enteric nervous system ganglia (Neunlist et  al. 1999; Obaid et al. 1999; Vanden et al. 2001; Schemann et al. 2002). Given the enormous potential of optical recording with fVSDs for studying neural networks, why have not more laboratories adopted this approach? A key reason is that the optical signals corresponding to action potentials in individual neurons are miniscule – often ranging from 0.001 to less than 0.0001 of the resting light level – making their detection quite challenging. Another reason is the difficulty of combining high resolution imaging with intracellular recording from multiple neurons. Although such combined methodology would be highly useful for circuit mapping, the light-efficient compound microscopes used for imaging lack stereopsis, making integrating multiple intracellular electrodes extremely difficult. Ideally, one could penetrate and drive a known neuron, image its followers, and then penetrate those followers with a second electrode to test for a direct synaptic connection. A third difficulty with using optical recording for network studies is the large, unwieldy data sets generated. Individual detectors of either camera or photodiode array systems often record multiple

neurons, yielding mixed signals. Conversely, multiple detectors often record the same neuron, yielding redundant traces. As a result, it can be very difficult to know the number of neurons included in the optical data set. Fortunately, it is possible to overcome all these difficulties. Here we describe procedures for obtaining satisfactory signal-to-noise ratio using fVSDs, for easier integration of sharp electrodes into imaging experiments, and for the transformation of raw data sets of mixed and redundant traces into new sets containing a single neuron per trace. This discussion is particularly relevant for investigators considering large-scale optical recording with fVSDs with single cell resolution, such as is readily achievable in invertebrate ganglia and in certain vertebrate peripheral nervous system preparations. Here we focus primarily on the use of photodiode arrays.

5.2  Choice of Fluorescence Vs. Absorbance Fast Voltage Sensitive Dyes Fast voltage-sensitive dyes come in two main types: absorbance and fluorescence (Ebner and Chen 1995; Zochowski et al. 2000b). Both change their light response linearly with membrane potential, and do so fast enough to trace out each action potential. Generally speaking, absorbance dyes have been preferred for network studies, in part due to their lower toxicity at the high light levels needed to attain sufficient signal-to-noise ratio. Direct comparisons in the vertebrate brain slice have found absorbance dyes to yield larger signal-tonoise with less phototoxicity than the tested fluorescence dyes (Jin et al. 2002; Chang and Jackson 2003). Two such absorbance dyes, RH155 and RH482, have been applied to preparations ranging from invertebrate ganglia (Yagodin et  al. 1999), to vertebrate slice (Senseman 1996; Momose-Sato et al. 1999; Yang et al. 2000), to cell culture preparations (Parsons et al. 1991). We have similarly used both dyes successfully in Tritonia and Aplysia. Recently, fast fluorescent dyes have been developed and used that have lower phototoxicity and other desired qualities than previous dyes (for example, see Obaid et  al. 2004; Carlson and Coulter 2008). We have not yet tried these on our invertebrate preparations. Recently developed calcium dyes also allow imaging of the firing of hundreds of individual neurons simultaneously in the

William N. Frost, Jean Wang, Christopher J. Brandon and Evan S. Hill  •  Department of Cell Biology and Anatomy, The Chicago Medical School, Rosalind Franklin University of Medicine and Science, North Chicago, IL 60064, USA Caroline Moore-Kochlacs and Terrence J. Sejnowski  •  Howard Hughes Medical Institute, The Salk Institute for Biological Studies, La Jolla, CA 92037, USA Terrence J. Sejnowski  •  Division of Biological Sciences, University of California San Diego, La Jolla, CA 92093, USA M. Canepari and D. Zecevic (eds.), Membrane Potential Imaging in the Nervous System: Methods and Applications, DOI 10.1007/978-1-4419-6558-5_5, © Springer Science+Business Media, LLC 2010

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the slow dynamics of calcium resequestration means that action potentials occurring in trains and bursts merge into a single waveform. Fast VSDs thus remain superior for network studies requiring single action potential resolution in multiple neurons.

5.3  Choice of Imaging System

FIGURE 5.1.  Imaging with fast voltage sensitive dyes allows simultaneous recording of the individual action potentials generated by large numbers of neurons. (A) Conventional sharp electrode intracellular recording of the Tritonia swim motor program. A 1 s 10 Hz stimulus to Pedal Nerve 3 triggered a 5 cycle swim motor program, recorded in the C2 and DSI interneurons of the swim central pattern generator. Experiments using sharp electrodes are typically limited to recording 3–4 neurons simultaneously. (B) Optical recording of the Tritonia swim motor program, after processing with Independent Component Analysis, showing 41 of the 63 neurons detected. A 2 s 10 Hz stimulus to Pedal Nerve 3 was used to trigger this 5 cycle motor program, which was recorded with a RedShirtImaging NeuroPDA-III 464 photodiode array and the fast absorbance voltage sensitive dye RH155.

vertebrate brain in  vivo (Stosiek et  al. 2003; Kerr et  al. 2005; Sato et  al. 2007; Takahashi et  al. 2007; Greenberg et  al. 2008). Although these dyes can resolve isolated single action potentials,

Most imaging systems employ either a camera or photodiode array. Both designs operate as a spatial array of independent sensors that convert light to voltage, with the entire array sampled at a fixed frequency. Two systems we have tried by RedShirtImaging are the NeuroCMOS-SM128 camera and the NeuroPDA-III photodiode array. The CMOS-SM128 has 16,384 independent sensors arranged on a 128 × 128 chip, with the full set sampled at 2,500 Hz. Using the RedShirtImaging Neuroplex software, the data can be viewed either as a camera image of the preparation at different points in time, with pixel color or grayscale brightness indicating the membrane potential of that location, or as a set of up to 16,384 voltage traces (one trace per pixel). The latter can be spatially averaged using either grid-based binning or user-outlined regions of interest, such as single neurons. By contrast, the RedShirtImaging NeuroPDA-III is organized as a hexagonal array of 464 sensors (Fig. 5.2), with the full set sampled at 1,600 Hz. The PDA data are shown as miniature traces organized on screen in the positions of the diodes that recorded them. Because the PDA does not have the spatial resolution to form a useful image, the 464 miniature traces are superimposed on an image of the preparation taken by a separate digital camera that can also be used to focus the PDA on the neurons of interest. As with the camera system, diodes can be selected individually or in groups for display of their recorded voltage traces, raw or filtered, individual or spatially averaged, in a separate window in Neuroplex (Fig. 5.3). Cameras and photodiode arrays have different strengths and weaknesses. Cameras such as the CMOS-SM128 have sufficient pixel density to provide a recognizable image, which simplifies focusing the imager and determining which regions and/or neurons gave rise to the recorded signals. On the negative side, the camera’s larger sensor number leads to filesizes approximately 50× larger than those acquired by the PDA for the same recording duration. Another difference regards the sensitivity of the two types of systems. While both systems digitize the data with 14 bit resolution (the CMOS system in the camera, the PDA via an A/D board in the computer), the NeuroPDA-III gains nearly seven additional bits of effective resolution by first AC-coupling each trace to subtract the resting light level, and then amplifying it by 100× before digitization. This makes PDAs more suitable for detecting the smaller signals provided by absorbance dyes if digitization resolution is a limiting factor, a point made by several others (Sinha and Saggau 1999; Vanden Berghe et al. 2001; Kosmidis et al. 2005). It is worth noting, however, that AC-coupled PDAs are unsuited for studies of slow events, such as slow EPSPs. The majority of our remaining discussion concerns our experience with the NeuroPDA-III.

5.4  Two Microscope Designs for Optical Recording We use two different microscope designs, depending on the degree to which intracellular electrodes will be used in the experiment. In both systems (Fig. 5.4), the microscope is mounted on an EXFOBurleigh motorized translator attached to a Burleigh Gibraltar stage

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FIGURE 5.2.  How the RedshirtImaging NeuroPDA-III sees and displays the data. (A) Hexagonal face formed by the ends of the bundle of 464 fiber optics upon which the image of the preparation is focused. The other ends of these fiber optics are individually glued to their corresponding diodes of the 464 photodiode array. (B) Depiction of an RH155-stained Tritonia pedal ganglion focused onto the array input port. Individual neurons of many different sizes are evident. The largest neurons will be recorded by up to several dozen photodiodes, leading to redundancy in the recordings, while the smallest will be detected by just one or two photodiodes. (C) Data display in Neuroplex. As soon as the data are acquired, Neuroplex displays them as miniature traces organized according to the position of each acquiring diode in the array. Shown is a 35 s optical recording of a swim motor program, obtained from a different preparation than the one photographed in panel (B). Nearly 50 diodes detected a single large neuron located in the upper right portion of the array. Clicking on specific traces expands them in a separate window in Neuroplex (see Fig. 5.3).

FIGURE 5.3.  Superimposing an image of the preparation with the diode array makes it easy to inspect the firing of different neurons. The left panel shows an image of the dorsal surface of the pedal ganglion, superimposed in Neuroplex with a display of the 464 acquired optical traces in their corresponding diode positions (green lines). The right panel shows how clicking on diodes of interest displays their optical data. These are filtered, unaveraged recordings from single diodes, each showing the firing recorded from the indicated ganglion location. A five cycle swim motor program was elicited by a 10 Hz, 1 s, 10 V stimulus to Pedal Nerve 3, delivered at the arrow.

that provides a large stable platform for mounting the manipulators, recording chamber, suction electrodes, bath ground, bath temperature probe, optional epi-illumination, perfusion tubes, and cooling system used in our Tritonia experiments. The translator allows penetration of a neuron in one ganglion, and then subsequent movement of the microscope and PDA without disturbing the impalement, in order to optically record from neurons in other ganglia that are affected by spike trains driven in the impaled neuron.

5.4.1  Conventional Compound Microscope Once vibration-related noise is sufficiently minimized, the signalto-noise ratio is proportional to the square root of the light level (Zochowski et al. 2000b). Therefore, imaging is traditionally done using conventional compound microscopes (Fig. 5.4A), whose high NA objectives are much more light-efficient than those of stereomicroscopes. We use an Olympus BX51WI microscope, which was designed specifically for electrophysiology. Our lens of

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FIGURE 5.4.  Two microscope designs used for imaging. (A) For experiments involving minimal-to-no conventional intracellular recording we use a standard compound microscope. A parfocal digital camera is used to focus the PDA, and to provide a picture of the preparation to superimpose with the array data. (B) For experiments involving significant intracellular recording with the imaging we use a custom microscope consisting of the lower half of a compound microscope, to provide the bright transillumination needed for absorbance fVSDs, mated to and optically aligned with a stereomicroscope. A linear sliding lens switcher is used to alternate between a 0.6 NA 20× objective for the imaging, and a parfocal stereomicroscope objective for penetrating neurons with sharp electrodes. During experiments the imaging lens is fitted with a custom water immersion cap to eliminate image movement related to surface ripples. Both microscopes are mounted on motorized translators to allow the viewing/imaging area to be changed without disturbing impaled neurons. During experiments, the upper superstructure is further stabilized by inserting two foam-topped posts between the Gibraltar platform and the front corners of the lens switcher. A digital eyepiece camera is used with this microscope to obtain the images of the preparation to be superimposed with the array data.

choice is the Olympus XLUMPLFL20×W 0.95 NA water immersion objective. In Tritonia ganglia transilluminated with a 100 W, 12 V tungsten-halogen lamphouse and 0.9 NA condenser, this lens provides saturating light levels to the PDA with the lamp power set to slightly above half-maximum. High efficiency optics are important because they permit lower illumination intensities, translating to slower dye bleaching, lower phototoxicity concerns, and longer imaging time. With this microscope and optics we can obtain several 60 s acquisitions at near-saturating light levels for the PDA before dye bleaching begins to degrade the action potential signals.

5.4.2  Custom Hybrid Microscope Despite their advantages, conventional compound microscopes lack stereopsis, making it difficult to incorporate multiple intracellular electrodes into imaging experiments. This is a major hindrance for many potential uses of optical recording, such as circuit mapping, where one would like to locate and immediately penetrate specific neurons identified in the optical recordings. Another aim is to drive a known neuron with one intracellular electrode while imaging its follower neurons, and then penetrate those with a second intracellular electrode. To carry out such experiments, we constructed a hybrid microscope that combines desirable features of both stereo and conventional compound microscopes (Fig. 5.4B).

We began by cutting off the top of a Nikon Optiphot microscope and then mounted a Zeiss SV-11 stereomicroscope onto a vertical post bolted to its stump. This allows the preparation to be viewed through the stereo objective, with the full depth perception necessary to penetrate specific neurons with intracellular electrodes. At the same time, we retained the compound microscope’s lamphouse and condenser, to deliver maximum light to the preparation for imaging. A key part of this design is the linear sliding lens switcher (Combizoom 400, Kramer Scientific), which allows the user to alternate between a high NA objective for imaging and a conventional parfocal stereomicroscope objective for impaling neurons. This hybrid microscope design has been described in detail in a separate publication (Frost et al. 2007). While the ability to integrate multiple intracellular electrodes into optical recording experiments is a big plus, the hybrid microscope has certain disadvantages compared with the traditional compound microscope. First, light transmission is lower through the zoom optics of the stereomicroscope body, requiring delivery of brighter illumination to the preparation to achieve adequate signal-to-noise ratios. To get sufficient illumination, we power a traditional 12 V 100 W tungsten halogen bulb with a 16 V racing car battery stepped down to 14.5 V. This has the advantage of true DC power, and hence no ripple in the light to the imager. However, the brighter light comes at the cost of faster photobleaching and thus less imaging time per experiment (four to five 35 s acquisitions vs. several 60 s acquisitions with the conventional compound microscope setup). Second, the rear post mount of the stereomicroscope and PDA leads to increased vibration-based noise in the optical recordings. We minimize the vibration by inserting support posts beneath the front of the lens switcher, effectively supporting the microscope in three places. This design has greatly facilitated our use of multiple intracellular electrodes during imaging experiments. With intracellular recording comes the need to expose preparations to light while positioning electrodes and penetrating neurons. To avoid bleaching the fVSD while working with intracellular electrodes, we illuminate the preparation through a green filter that blocks the red wavelengths used for imaging with RH155.

5.5  Correlating Optical Signals with Specific Neurons in the Preparation Because of its poor spatial resolution, a PDA doesn’t provide a useful image – its frame view mode looks like a hexagonal checkerboard of grayscale squares, corresponding to the brightness recorded by the different diodes at one point in time. Therefore, identifying the neurons the data are coming from is more difficult with a PDA than with camera-based imagers, which have higher spatial resolution. To do this, we take a picture of the preparation and superimpose it in Neuroplex with a diode map linked to the optical data. Clicking on any diode/neuron then displays the optical data recorded at that location (Fig. 5.3). The image superimposition involves a three-step procedure.

5.5.1  S et the Camera and PDA Parfocal with One Another During experiments with the PDA, the focus point for the imaging is set using the live image provided by the microscope camera used to take the superimposed photograph. To insure that the PDA and camera are parfocal, we focus the camera on a calibration slide containing a black circle, and then adjust the focusing collar under the PDA so that the circle’s edge shows the most abrupt

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black-to-white transition on the checkerboard pattern provided by the PDA. An alternative focusing method is to remove the PDA from its photoport and place a ground glass surface at the exact position the array face normally sits. Adjustments are then made to bring this image and that seen by the camera into parfocality. A third method is suggested by (Obaid et  al. 2004), who used a custom microscope photoport component which, when slid into place, provides a direct view of the preparation image focused onto the photodiode array face.

5.5.2  O  btain a Single, In-Focus Picture to Superimpose on the Array Data For three-dimensional neuronal preparations such as invertebrate ganglia, a single image taken through the objective lens will typically have just a portion of the surface neurons in focus. To obtain a clear picture of the entire visible ganglion surface to superimpose with the PDA data we take a stack of several images at different focal depths and then combine them into a single in-focus image (Fig. 5.5) using the fast focus enhancement feature of Rincon (ImagingPlanet, Goleta, CA).

5.5.3  A  ccurately Align the Camera Picture with the PDA Data To correlate the imaging data with specific neurons, the above photograph must be precisely superimposed with the diode map. To do this, we collect both a camera picture and PDA readings of the light shining through three closely spaced pinholes in a foil sheet. The magnification and position of the camera image is then adjusted in Neuroplex until it exactly overlies the PDA image of the pinholes, rotating the PDA as needed, after which it is locked into position. In subsequent experiments these same coordinates can then be used to accurately superimpose the camera image of the preparation on the array data.

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5.6  Getting Good Signals When recording with fVSDs, neuronal action potentials may produce signals in the range of 0.001 to less than 0.0001 of the resting light level. Discriminating such small signals is a challenge. Here we describe some key steps that make this possible using the absorbance dye RH155 in the marine mollusks Tritonia diomedea and Aplysia californica.

5.6.1  Choose an Optimal Transmission Filter The ability to see the small signals provided by voltage sensitive absorption dyes requires the use of bandpass filters optimized for each dye. RH155 and RH482, for example, have biphasic absorption curves – below a certain wavelength they decrease their absorption with depolarization, above it they increase absorption (Parsons et al. 1991; Jin et al. 2002). To avoid signal cancellation, the bandpass filter selected for use with such dyes must thus be centered on one side or the other of the crossover wavelength, with an appropriately limited bandwith. After trying several, we chose a 725/25 bandpass filter (Chroma Technology) for our work with Tritonia and Aplysia using RH155. Because the spectral characteristics of voltage sensitive dyes can shift in different solutions and tissues, the filter properties needed to provide optimal neuronal signals should be determined in the preparation of interest (Wang et al. 2009).

5.6.2  G  et the Light as Close to Saturation as Possible Once vibration-related noise has been sufficiently minimized (see below), signal-to-noise ratio increases in proportion to the square root of the illumination intensity (Zochowski et  al. 2000b). This means that optimal optical recordings will be obtained when the resting light level reaching the imager is as close to the device’s saturation level as possible. There are two steps for achieving this:

FIGURE 5.5.  Use of software to obtain a single in-focus image to superimpose with the photodiode array data. To obtain a suitable picture of the highly convex Tritonia ganglion surface to superimpose with the array data in Neuroplex during experiments, we use software that combines the in-focus portions of several images taken at different focus positions, while discarding the blurred areas. (A) Uppermost surface of the sheathed Tritonia dorsal pedal ganglion. (B) Focused 100 mm lower. (C) 200 mm lower. (D) 300 mm lower. (E) Combination of 13 images taken at 25 mm intervals, showing all neurons visible on the ganglion surface.

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(1) maximize the light to the preparation, and (2) maximize the light transmitted from the preparation to the imager.

5.6.2.1  M  aximize the Light to the Preparation

The lamphouse should be adjusted to position the bulb filament directly in the center of the field of view. If it has an adjustable back mirror, this should be positioned to make the image as bright as possible. Built-in diffusion and heat filters, the latter which attenuates the wavelength we use with RH155 (725 nm), can be removed from the light path between the lamphouse and the condenser to maximize light transmission. Alternatively, the built-in heat filter could be replaced with one chosen to block IR wavelengths while passing those needed for the particular dye being used. Different condensers concentrate the light into smaller or larger areas, reflecting the magnification range of the objectives they were designed to work with. The typical condenser illuminates an area of the recording chamber far larger than the area seen by the 20× objective we use in most experiments. After trying several condensers, the brightest light delivery so far was obtained with a simple 0.9 NA Abbe achromat condenser having a swing-in top lens. This style is designed for use with low magnification objectives with the top lens removed, and higher magnification lenses with the top lens in place, which concentrates the light into a smaller area, closer to the field of view of our 20× imaging objective. The condenser height is empirically adjusted to produce the brightest illumination of the preparation, which occurs near the point of Kohler illumination, when the base diaphragm of the microscope is in sharp focus. With our preferred condenser, this occurs close enough to the preparation to require a recording chamber having a cover slip bottom topped by a layer of Sylgard just thick enough to hold the minute pins used to position the preparation. Under Kohler illumination, the microscope base diaphragm can be adjusted to constrict the illuminated area to the exact area being imaged. This prevents bleaching of other areas in the preparation, such as nearby ganglia, allowing them to be imaged later in the experiment.

5.6.2.2  M  aximize the Light from the Preparation to the Imager

Increased efficiency in this part of the light path allows the desired near-saturating light levels to be obtained with lower lamp brightness, and thus slower dye bleaching and reduced potential phototoxicity. It also translates to longer imaging time, beneficial for monitoring long duration events such as the Tritonia swim motor program. An important way to maximize light transmission in this part of the pathway is to use the highest NA aperture objective lens available for the desired magnification, matched with a condenser with a comparable NA. A photoport that allows 100% of the light to be directed to the imager should also be used.

5.6.3  Reduce Vibration-Related Noise The major source of noise in PDA experiments arises from vibration, which causes small oscillatory movements of contrast edges in the preparation on the photodiodes, producing oscillations in the recordings that can be many times the size of the optically recorded action potentials. Common sources of vibration include the opening of the shutter controlling the light to the preparation, pulsations related to perfusion pumps, and movements of the floor and walls caused by the building air handling systems, as well as by the movements of people in and outside the laboratory. A vibration isolation table is essential for minimizing externally-originating vibrations.

In addition, we (1) work on the ground floor, (2) use a compound microscope designed with low vibration and electrophysiology in mind (Olympus BX51WI), (3) use commercial water immersion objectives, or affix custom-made water immersion caps to our non-water immersion objectives to eliminate vibrations at the air/ solution interface, (4) mount the imaging shutter on the frame of the air table rather than on its surface, (5) ensure that cables connected to outside devices contact the table before reaching the microscope and imager, and (6) for the hybrid microscope, which otherwise would be supported only by the back mounting post, we insert two metal posts topped with foam pads between the frame of the lens switcher and the Gibraltar stage. The Neuroplex software can readily reveal the effects of these vibration-reducing efforts by displaying a Fourier analysis of the frequency components in the optical recordings.

5.6.4  I ssues Related to Preparations Requiring Cold Saline For preparations requiring cold saline, such as Tritonia (11°C), condensation can form on the bottom side of the recording chamber, interfering with imaging. To prevent this, we place a drop of fluid on the condenser lens, which then fuses to the bottom of the recording chamber when the condenser is raised to its working position. In times of high humidity, significant condensation can also form on the inside of water immersion objective lenses, or on the inside of custom-made water immersion lens caps. During such conditions, we run a dehumidifier in the laboratory, and also place the objective and any lens cap in a dessicant-containing sealed plastic bag the night before the experiment. Just before recording, we attach the cap within the bag before placing the lens on the microscope. Our custom lens cap has a lightly greased O-ring within it to both hold it securely on the lens and to establish an airtight seal.

5.6.5  O  ther Methods for Increasing the Number of Neurons Detected in the Imaging Although high-NA objective lenses are highly desirable for their light efficiency, this comes at the cost of a narrower depth of focus. This can be advantageous – the focus point of the microscope provides information on the depth location of the optically recorded neurons. A disadvantage is that signal loss from out-of-focus neurons means that many are not detected. A simple method to increase the number of neurons detected optically is to gently flatten the ganglion using a cover slip fragment held in place by Vaseline placed beside the ganglion on the chamber floor. In our experience, this simple procedure can double the number of neurons recorded optically.

5.7  Spike-sorting the Raw Optical Data with Independent Component Analysis Using the above techniques, we routinely obtain simultaneous optical recordings of several dozen to well over 100 neurons from the dorsal surface of the Tritonia pedal ganglion. Many scientific issues can be addressed by simply inspecting the filtered, but otherwise, raw diode recordings. For circuit mapping, previously unknown neurons that fire during a given motor program are easily identified. Neurons excited or inhibited by neurons driven with intracellular electrodes are also easily spotted. Other issues, however, cannot readily be addressed through simple visual inspection of the data traces. For example, it is nearly impossible to determine

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FIGURE 5.6.  Use of Independent Component Analysis to sort the raw data into a set of single-neuron traces. (A) Selected traces from the raw diode data, after processing with 5 Hz high-pass and 100 Hz low-pass Butterworth software filters in Neuroplex. A 10 Hz, 2 s stimulus was applied to Pedal Nerve 3 at the arrow, eliciting a 4 cycle swim motor program. The raw traces contain redundantly recorded neurons (see the large action potentials in traces 1 and 2, and 11 and 12, as well as mixtures of more than one neuron per trace (most apparent in traces 2, 9, 10, and 11). Although neurons of interest are readily identified in the raw data, it is impossible to determine how many neurons were recorded by visual inspection alone. (B1) Selected traces of the 61 components containing neurons that were extracted by ICA from the 464 diode raw data set. ICA transforms the recorded set of signal mixtures into their original single-neuron sources. The top trace is the independent component corresponding to the neuron with large action potentials recorded redundantly by the top two diodes in panel (A). The bottom trace is the independent component corresponding to the neuron recorded redundantly by the bottom two diodes in panel (A). ICA is an automated process that can be run on any Matlab-equipped personal computer. Note that the prominent artifact present shortly after the stimulus in most of the raw data traces is absent from the component traces, having been extracted as a separate independent component (not shown). (B2) Array maps of the diodes contributing to three components, indicating the XY position and approximate size of the source neurons.

the number of neurons present in a typical optical recording. The larger neurons appear redundantly in many traces, while many traces contain action potentials from multiple neurons (Fig. 5.6A). To take full advantage of the power of optical recording, we need a method for spike sorting these mixed and redundant data sets. For several years beginning in the late 1980s, Larry Cohen and colleagues applied a spike template matching method for sorting the redundant and mixed raw spike data into a new set of traces consisting of one unique neuron per trace (Zecevic et al. 1989; Falk et  al. 1993; Tsau et  al. 1994; Wu et  al. 1994a, Wu et  al. 1994b; Hickie et  al. 1997; Zochowski et  al. 2000a). A downside of this method was its heavy reliance on experimenter judgment, leading to a typical processing time of several days per record (Cohen et al. 1989). More recently, Independent Component Analysis (ICA) has been introduced as a rapid and automated method for spike-sorting optical recording data sets obtained with fVSDs (Brown et al. 2001; Brown et al. 2008). ICA is a blind source separation methodology for separating signal mixtures into their original sources. ICA finds a linear transformation of the raw traces that minimizes the mutual information shared between the output components, separating the redundant and mixed neuronal source signals into separate, single-neuron traces (Fig. 5.6B1). For an imager with N pixels or diodes, ICA can separate up to N independent components, which can include artifacts and other noise sources, effectively removing them from the neuronal recordings (Brown et al. 2001). We used infomax ICA (Bell and Sejnowski 1995; Delorme and Makeig 2004). With a HP Xeon 3.2 GHz Z400 workstation with 8 GB RAM running Windows XP Professional x64, ICA can process a 35 s file of 464 optical traces digitized at 1,600 Hz into the set of independent components in as little as 9 min. MATLAB

programs have also been written (C.M.-K.) that return maps showing the position of each resolved component on the diode array (Fig. 5.6B2). Using ICA we are able to (1) eliminate redundancy by combining neurons recorded by multiple diodes into single traces, (2) sort multiple neurons recorded by single diodes into separate singleneuron traces, (3) provide the array location of each resolved neuron, and (4) remove artifacts from the traces. ICA thus represents a powerful, fast, and automated tool for spike sorting data sets obtained with photodiode arrays and fast voltage sensitive dyes.

Acknowledgments Supported by NS060921, Dart Foundation and Grass Foundation Marine Biological Laboratory summer fellowships, the Fred B. Snite Foundation, Rosalind Franklin University of Medicine and Science (WF), and the Howard Hughes Medical Institute (TS). We thank JY Wu, LB Cohen and L Eliot for comments on the manuscript.

References Bell AJ, Sejnowski TJ (1995) An information-maximization approach to blind separation and blind deconvolution. Neural Comput 7:1129–1159. Boyle MB, Cohen LB, Macagno ER, Orbach H (1983) The number and size of neurons in the CNS of gastropod molluscs and their suitability for optical recording of activity. Brain Res 266:305–317. Brown GD, Yamada S, Sejnowski TJ (2001) Independent component analysis at the neural cocktail party. Trends Neurosci 24:54–63.

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Brown GD, Yamada S, Nakashima M, Moore-Kochlacs C, Sejnowski TJ (2008) Independent component analysis of optical recordings from Tritonia swimming neurons. In: Technical Report INC-08-001, Institute for Neural Computation, University of California at San Diego. Carlson GC, Coulter DA (2008) In vitro functional imaging in brain slices using fast voltage-sensitive dye imaging combined with whole-cell patch recording. Nat Protoc 3:249–255. Chang PY, Jackson MB (2003) Interpretation and optimization of absorbance and fluorescence signals from voltage-sensitive dyes. J Membr Biol 196:105–116. Cohen L, Hopp HP, Wu JY, Xiao C, London J (1989) Optical measurement of action potential activity in invertebrate ganglia. Annu Rev Physiol 51:527–541. Delorme A, Makeig S (2004) EEGLAB: an open source toolbox for analysis of single-trial EEG dynamics including independent component analysis. J Neurosci Methods 134:9–21. Ebner TJ, Chen G (1995) Use of voltage-sensitive dyes and optical recordings in the central nervous system. Prog Neurobiol 46:463–506. Falk CX, Wu J, Cohen LB, Tang AC (1993) Nonuniform expression of habituation in the activity of distinct classes of neurons in the Aplysia abdominal ganglion. J Neurosci 13:4072–4081. Frost WN, Wang J, Brandon CJ (2007) A stereo-compound hybrid microscope for combined intracellular and optical recording of invertebrate neural network activity. J Neurosci Methods 162:148–154. Greenberg DS, Houweling AR, Kerr JN (2008) Population imaging of ongoing neuronal activity in the visual cortex of awake rats. Nat Neurosci 11:749–751. Grinvald A, Salzberg BM, Cohen LB (1977) Simultaneous recording from ­several neurones in an invertebrate central nervous system. Nature (Lond) 268:140–142. Hickie C, Cohen LB, Balaban PM (1997) The synapse between LE sensory neurons and gill motoneurons makes only a small contribution to the Aplysia gill-withdrawal reflex. Eur J Neurosci 9:627–636. Jin W, Zhang RJ, Wu JY (2002) Voltage-sensitive dye imaging of population neuronal activity in cortical tissue. J Neurosci Methods 115:13–27. Kerr JN, Greenberg D, Helmchen F (2005) Imaging input and output of neocortical networks in vivo. Proc Natl Acad Sci U S A 102:14063–14068. Kojima S, Hosono T, Fujito Y, Ito E (2001) Optical detection of neuromodulatory effects of conditioned taste aversion in the pond snail Lymnaea stagnalis. J Neurobiol 49:118–128. Kosmidis EK, Cohen LB, Falk CX, Wu JY, Baker BJ (2005) Imaging with voltage-sensitive dyes: spike signals, population signals, and retrograde transport. In: Yuste R, Konnerth A (eds) Imaging in neuroscience and development. A laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. London JA, Zecevic D, Cohen LB (1987) Simultaneous optical recording of activity from many neurons during feeding in Navanax. J Neurosci 7:649–661. Momose-Sato Y, Sato K et al. (1999) Evaluation of voltage-sensitive dyes for long-term recording of neural activity in the hippocampus. J Membr Biol 172:145–157. Nakashima M, Yamada S, Shiono S, Maeda M, Satoh F (1992) 448-Detector optical recording system: development and application to Aplysia gillwithdrawal reflex. IEEE Trans Biomed Eng 39:26–36. Neunlist M, Peters S, Schemann M (1999) Multisite optical recording of excitabi­ lity in the enteric nervous system. Neurogastroenterol Motil 11:393–402. Nikitin ES, Balaban PM (2000) Optical recording of odor-evoked responses in the olfactory brain of the naive and aversively trained terrestrial snails. Learn Membr 7:422–432. Obaid AL, Koyano T, Lindstrom J, Sakai T, Salzberg BM (1999) Spatiotemporal patterns of activity in an intact mammalian network with single-cell

resolution: optical studies of nicotinic activity in an enteric plexus. J Neurosci 19:3073–3093. Obaid AL, Loew LM, Wuskell JP, Salzberg BM (2004) Novel naphthylstyrylpyridium potentiometric dyes offer advantages for neural network analysis. J Neurosci Methods 134:179–190. Parsons TD, Salzberg BM, Obaid AL, Raccuia-Behling F, Kleinfeld D (1991) Long-term optical recording of patterns of electrical activity in ensembles of cultured Aplysia neurons. J Neurophysiol 66:316–333. Salzberg BM, Grinvald A, Cohen LB, Davila HV, Ross WN (1977) Optical recording of neuronal activity in an invertebrate central nervous system: simultaneous monitoring of several neurons. J Neurophysiol 40:1281–1291. Sato TR, Gray NW, Mainen ZF, Svoboda K (2007) The functional microarchitecture of the mouse barrel cortex. PLoS Biol 5:e189. Schemann M, Michel K, Peters S, Bischoff SC, Neunlist M (2002) Cuttingedge technology. III. Imaging and the gastrointestinal tract: mapping the human enteric nervous system. Am J Physiol Gastrointest Liver Physiol 282:G919–G925. Senseman DM (1996) High-speed optical imaging of afferent flow through rat olfactory bulb slices: voltage-sensitive dye signals reveal periglomerular cell activity. J Neurosci 16:313–324. Sinha SR, Saggau P (1999) Optical recording from populations of neurons in brain slices. In: Johanson H, Windhorst U (eds) Modern techniques in neuroscience research. Springer Verlag, Berlin. Stosiek C, Garaschuk O, Holthoff K, Konnerth A (2003) In vivo two-photon calcium imaging of neuronal networks. Proc Natl Acad Sci U S A 100: 7319–7324. Takahashi N, Sasaki T, Usami A, Matsuki N, Ikegaya Y (2007) Watching neuronal circuit dynamics through functional multineuron calcium imaging (fMCI). Neurosci Res 58:219–225. Tsau Y, Wu J, Hopp H, Cohen LB, Schiminovich D, Falk CX. (1994) Distributed aspects of the response to siphon touch in Aplysia: spread of stimulus information and cross-correlation analysis. J Neurosci 14:4167–4184. Vanden Berghe P, Bisschops R, Tack J (2001) Imaging of neuronal activity in the gut. Curr Opin Pharmacol 1:563–567. Wang Y, Jing G, Perry S, Bartoli F, Tatic-Lucic S (2009) Spectral characterization of the voltage-sensitive dye di-4-ANEPPDHQ applied to probing live primary and immortalized neurons. Opt Express 17:984–990. Wu J, Cohen LB, Falk CX (1994a) Neuronal activity during different behaviors in Aplysia: a distributed organization? Science 263:820–823. Wu J, Tsau Y, Hopp H, Cohen LB, Tang AC, Falk CX (1994b) Consistency in nervous systems: trial-to-trial and animal-to-animal variations in the responses to repeated applications of a sensory stimulus in Aplysia. J Neurosci 14: 1366–1384. Yagodin S, Collin C, Alkon DL, Sheppard NF Jr, Sattelle DB (1999) Mapping membrane potential transients in crayfish (Procambarus clarkii) optic lobe neuropils with voltage-sensitive dyes. J Neurophysiol 81:334–344. Yang S, Doi T, Asako M, Matsumoto-Ono A, Kaneko T, Yamashita T (2000) Multiple-site optical recording of mouse brainstem evoked by vestibulocochlear nerve stimulation. Brain Res 877:95–100. Yuste R (2008) Circuit neuroscience: the road ahead. Front Neurosci 2:6–9. Zecevic D, Wu J, Cohen LB, London JA, Hopp H, Falk CX (1989) Hundreds of neurons in the Aplysia abdominal gang­lion are active during the gill-withdrawal reflex. J Neurosci 9:3681–3689. Zochowski M, Cohen LB, Fuhrmann G, Kleinfeld D (2000a) Distributed and partially separate pools of neurons are correlated with two different components of the gill-withdrawal reflex in Aplysia. J Neurosci 20:8485–8492. Zochowski M, Wachowiak M, Falk CX, Cohen LB, Lam YW, Antic S, Zecevic D (2000b) Imaging membrane potential with voltage-sensitive dyes. Biol Bull 198:1–21.

6

Monitoring Integrated Activity of Individual Neurons Using FRET-Based Voltage-Sensitive Dyes Kevin L Briggman, William B. Kristan, Jesús E. González, David Kleinfeld, and Roger Y. Tsien

6.1  Introduction Fluorescence resonance energy transfer (FRET) is a physical dipole– dipole coupling between the excited state of a donor fluorophore and an acceptor chromophore that causes relaxation of the donor to a non-fluorescent ground state, which excites fluorescence in the acceptor. Initially described by Förster (1948), FRET has been extensively reviewed (Stryer 1978; Clegg 1995; Selvin 2000; Lakowicz 2006). In practical terms, the efficiency of FRET depends on the properties of the chromophores and the distance between them, measured as the Förster radius (Ro): the distance between the donor and the acceptor at which half the energy is transferred. The magnitude of Ro depends on the donor quantum yield and the spectral overlap between the donor emission and the acceptor absorbance spectra. For commonly used synthetic FRET pairs, Ro values range from 2 to 8 nm (Wu and Brand 1994). FRET efficiency is inversely proportional to the sixth power of the donor and acceptor distance, providing a sensitive readout of intermolecular distances near Ro. Experimentally, FRET is measured either as the decrease in the lifetime or intensity of donor fluorescence after the addition of acceptor, or as the increase in acceptor fluorescence after the addition of donor. Because typical Ro values are similar to protein and membrane dimensions, FRET has proven useful in a wide variety of biochemical and cellular applications to investigate protein–protein interactions, protein and DNA conformational analysis, and membrane topography (Stryer 1978; Clegg 1995; Selvin 2000; Lakowicz 2006). Furthermore, with the advent of a large variety of fluorescent protein color variants, FRET has become a natural method to probe cellular biochemistry (Piston and Kremers 2007). Here, we review how FRET probes have been used to measure cellular membrane potentials. Voltage-sensitive dyes (VSDs) based upon FRET are composed of two molecules, with either the donor or acceptor being a hydrophobic anion introduced into the plasma membrane acting as the voltage sensor by translocating between the energy minima at the intracellular and extracellular membrane–water interfaces (Fig. 6.1A) (Gonzalez and Tsien 1995). When the transmembrane potential changes, the hydrophobic anion redistributes as an

e­ xponential function of the potential according to the Nernst ­equation. A second impermeant fluorophore is attached to one face of the membrane where it can undergo FRET with the mobile molecule, in proportion to the distance between the two molecules. When the impermeant fluorophore is bound to the extracellular membrane surface and the cell is at its normal (negative) resting potential, the anions are predominately near the extracellular face of the memb­rane, so that the two molecules produce efficient FRET. When the membrane depolarizes, for whatever reason, the anions equilibrate at a higher density at the intracellular membrane surface, thereby decreasing FRET. When both molecules are fluorescent, all increases in the acceptor emission are at the expense of the donor emission, and vice versa (Fig. 6.2A, B), thus providing a ratiometric signal of the membrane potential change. FRET-based dyes are sometimes called “slow” dyes, which is true relative to electrochromic dyes that have typical response times of a few microseconds (Ebner and Chen 1995; Baker et al. 2005), but FRET dye time constants actually span a large range, from 400 ms to 500 ms, depending on the properties of the mobile anion. While not the fastest, FRET dyes have yielded some of the largest observed fractional fluorescence changes, ranging from 10–20% per 100 mV in intact tissue (Cacciatore et al. 1999) to 100–300% per 100 mV in isolated cells (Gonzalez and Maher 2002). Following a brief account of the development of FRET VSDs, we discuss their temporal resolution, sensitivity, and phototoxicity. We then present examples of how these dyes have been used to image neurons. Finally, we provide detailed staining protocols as a reference and starting points for use in other systems.

6.2  Development of FRET Dye Pairs Gonzalez and Tsien (1995) were first to image voltage-dependent responses from a FRET-based VSD. They used negatively charged hydrophobic oxonol derivatives, bis-(1,3-dialkyl-2-thiobarbiturate)trimethineoxonol, DiSBACx(3), where x refers to the number of

Kevin L. Briggman  •  Department of Biomedical Optics, Max Planck Institute for Medical Research, Jahnstrasse 2969120, Heidelberg, Germany William B. Kristan  •  Neurobiology Section, Division of Biological Sciences, University of California, San Diego 9500 Gilman Drive, La Jolla, CA 92093-0357, USA Jesús E. González  •  6468 Wayfinders Court, Carlsbad, CA 92011, USA David Kleinfeld  •  Department of Physics, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0374, USA Roger Y. Tsien  •  Department of Pharmacology, Howard Hughes Medical Institute, University of California, San Diego 310 George Palade Labs, 9500 Gilman Drive, La Jolla, CA 92093-0647, USA M. Canepari and D. Zecevic (eds.), Membrane Potential Imaging in the Nervous System: Methods and Applications, DOI 10.1007/978-1-4419-6558-5_6, © Springer Science+Business Media, LLC 2010

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Chapter 6  •  K.L. Briggman et al.

FIGURE 6.1.  Properties of a FRET VSD dye pair. (A) Most FRET VSDs incorporate an immobile donor (purple) and a mobile anion (orange). A variety of molecules can be used for this purpose. Oxonol derivatives are a versatile choice for the mobile anion. When the donor fluorophore is excited (purple arrow), it will either emit blue photons when the mobile anion is near the intracellular surface of the membrane (blue arrow in the right diagram) or produce FRET when the mobile ion is near the extracellular surface (orange arrow in the left diagram). (B) The structure of a FRET donor, CC1-DMPE, a coumarin-labeled phospholipid. (C) The excitation and emission spectra for the CC2-DMPE and DiSBAC4(3) dye pair. The overlap between donor emission and acceptor excitation determines the FRET efficiency; the more complete the overlap, the more efficient is FRET. The emission spectra for these two dyes are well separated, making them a useful dye pair. (D) Emission spectra of the two dyes as DiSBAC4(3) was loaded into cell membranes. Increased concentrations of DiSBAC4(3) progressively quenched the CC2-DMPE emission. Panels (A) and (B) are reproduced with permission from Gonzalez and Tsien (1997), and panels (C) and (D) are reproduced with permission from Gonzalez and Maher (2002).

c­ arbons on the alkyl group and 3 refers to the number of polymethine carbons), as the mobile voltage sensor. Negatively charged oxonol molecules such as DiSBAC2(3) and bis-(1,3-dibutylbarbituric acid) trimethine oxonol [DiBAC4(3)] have previously been used as redistribution VSDs (Rink et al. 1980; Ebner and Chen 1995). They move between intracellular and extracellular compartments in response to membrane potential changes with very slow kinetics (seconds to minutes). An impermeant oxonol has also been used as a fast absorption dye (Grinvald et  al. 1981; Wu et  al. 1994b; Momose-Sato et al. 1999). Fluorescence changes result from the fact that oxonols are non-fluorescent in water but are highly fluorescent in hydrophobic environments such as membranes. An anion was chosen, rather than a cation, to take advantage of the dipole potential generated from the polar lipid head-groups, presumably from the ester carbonyl groups, which greatly speeds anion translocation. The rate differences for isostructural borates and phosphonium ions are orders of magnitude faster for the negatively charged borates (Flewelling and Hubbell 1986). Although the mobile anion can in principle serve as either the FRET donor or acceptor, it is most commonly used as the FRET acceptor. As discussed below, there are theoretical and practical advantages for the reverse configuration. The FRET donor was initially chosen to be fluoresceinlabeled wheat germ agglutinin (FL-WGA) that bound to N-acetylglucosamine groups on the extracellular face of plasma

membranes (Gonzalez and Tsien 1995). The same study showed that using longer chain alkyl groups not only increased the oxonol hydrophobicity but also resulted in faster membrane translocation speed, which improved the FRET temporal resolution, presumably by burying the oxonol deeper into the low dielectric region of the membrane. The optimal chain length was 6 and DiSBAC6(3) had a translocation time constant ~2 ms. The original dye pair was improved by replacing FL-WGA with a fluorescent phospholipid, N-(6-chloro-7-hydroxy-2-oxo2H-l-benzopyran-3-carboxamidoacetyl)-dimyristoylphosphatidyl ethanolamine [CC1-DMPE] (Gonzalez and Tsien 1997) (Fig. 6.1B). A second chlorocoumarin phospholipid CC2-DMPE is commercially available from Life Technologies (formerly Invitrogen); it has similar properties to CC1-DMPE. The coumarin has a high quantum yield (indistinguishable from 1) and makes an excellent FRET partner with trimethine thiobarbiturate oxonols. The fluorescence emission maxima are separated by 100 nm (Fig. 6.1C), thus enabling efficient collection of both donor and acceptor photons. Another key property of chlorocoumarin phospholipid donors is that they have two negative charges at physiological pH. This is important because it anchors the fluorescent head-group at the extracellular surface and prevents probe translocation across the plasma membrane which would greatly degrade the voltage-­ sensitive FRET signal. In addition to the phosphate charge, the

Monitoring Integrated Activity of Individual Neurons Using FRET-Based Voltage-Sensitive Dyes   •  Chapter 6

chloro-group reduces the pKa of the hydroxy coumarin (Fig. 6.1B) to ~5, which results in a second negative charge at physiological pH. A pyrene phospholipid has also been developed as a pH insensitive FRET donor that is compatible with low pH conditions used to activate acid-sensitive channels (Maher et al. 2007). The use of a phospholipid placed the donor fluorophore closer to the membrane– water interface, thereby decreasing the minimal distance between donor and acceptor, which may account for the increased voltage sensitivity with the fluorescent impermeant phospholipid (Gonzalez and Tsien 1997). In addition to the original oxonol dye DiSBAC6(3), longer wavelength pentamethine oxonols were developed (the oxonol shown in Fig. 6.1A has n = 2) (Gonzalez and Tsien 1997). Increasing the polymethine chain length from trimethine oxonol [DiSBAC6(3)] to pentamethine oxonol [DiSBAC6(5)] increases charge delocalization, thereby further lowering the ­activation energy for translocation. This change yielded a time ­constant for DiSBAC6(5) of ~0.4 ms. The FRET-based VSD strategy has more recently been used in hybrid voltage sensor systems, incorporating fast anions, such as dipicrylamine (DPA) or DiBAC4(5), with a genetically expressed donor fluorophore such as farnesylated enhanced GFP (eGFP-F) (Chanda et  al. 2005). DPA is an absorption dye that does not ­fluoresce; it serves only to quench the fluorescence of the donor mol-

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ecule when FRET occurs. The structure of the donor fluorophore has been additionally modified to be able to record action potentials (DiFranco et al. 2007; Sjulson and Miesenbock 2008). Together these developments and optimizations of FRET-based VSDs demonstrate the versatility in choosing the donor and acceptor for a variety of applications. Most importantly, the source of the voltage sensitivity is well understood making the rational design of future improvements possible.

6.3  Response Time of FRET Indicators Because the lifetime of FRET is short, on the order of nanoseconds, the time constant of FRET VSDs is determined by the rate that the hydrophobic anion equilibrates across the membrane when the membrane potential changes (Gonzalez and Tsien 1995). As mentioned above, increasing the length of the alkyl side chains of oxonol increases hydrophobicity and therefore increases its response speed. For example, DiSBAC2(3) and DiSBAC6(3) have time constants of 500 ms and 2 ms, respectively (Gonzalez and Maher 2002). However, increased hydrophobicity comes at the cost of reduced aqueous solubility, limiting loading concentrations (Table 6.1). Using pluronic F-127 and β-cyclodextrin brings the more hydrophobic oxonols into

FIGURE 6.2.  Voltage-dependent signals of FRET VSDs. (A) Leech neurons were voltage clamped and the membrane potentials was sinusoidally ­varied about a holding potential (bottom trace). The raw oxonol trace (top trace) was filtered (middle trace) to remove the slow bleaching artifact visible in the top trace. (B) Sequential recordings of the ­coumarin and oxonol emission wavelengths to the same sinusoidally varying intracellular stimulus ­demonstrating that, because the two dyes produce out-of-phase ­signals, the sensitivity can be enhanced by measuring the ratio of the two signals. (C) The sensitivity of the FRET signal is a function of the membrane potential measured at various holding potentials. The data are well fit by the expected redistribution of oxonol across the membrane during changes in membrane potential (open circles). FRET VSDs in leech ganglia are significantly more sensitive than other VSDs such as di-4-ANEPPS (filled circles). Panels reproduced with permission from Cacciatore et al. (1999).

TABLE 6.1.  Oxonol Derivatives that can be Used as the Mobile Anion and Their Associated Time Constants and Measured Sensitivities DiSBAC2(3) DiSBAC4(3) DiSBAC6(3) DiSBAC2(5) DiSBAC4(5) DiSBAC6(5)

Commercially Available

Pluronic Loading

λex (nm)

λem (nm)

Yes Yes No No No No

Optional Suggested Required Optional Suggested Required

540 540 540 640 640 640

560 560 560 660 660 660

Table reproduced with permission from Gonzalez and Maher (2002) a Not optimized

Tc (ms) 500 20 2 50 2 0.40

Vm sensitivity % ΔR per mV 1–3 0.6–1 0.4–0.8 0.5–2a 715

5.0E-002; 625/>715

5.4E-002; 630/>715

2.0E-001; 618/>715

1.2E-001; 610/>665

1PF Change (DF/F-100 mV) Ex/Em (nm)

8.09 ± 0.42

6.18 ± 0.68

11.64 ± 0.60

10.13 ± 0.88

10.70 ± 0.59

5.65 ± 0.45

1.82 ± 0.18

3.75 ± 0.33

9.56 ± 0.42

4.15 ± 0.28

3.55 ± 0.55

3.84 ± 0.38

SHG Change (%/50 mV)

−5.65 ± 0.50

6.72 ± 0.58

0.29 ± 0.39

−6.12 ± 0.87

−3.86 ± 0.53

−4.20 ± 0.13

−16.64 ± 0.85

−4.80 ± 0.53

−2.57 ± −0.24

−7.51 ± 0.73

−6.61 ± 0.88

0.79 ± 0.37

2PF Change (%, 615–665 nm/ 50 mV)

3.51 ± 0.66

–

8.78 ± 1.08

5.77 ± 1.22

6.52 ± 0.76

3.12 ± 0.18

−4.28 ± 0.54

2.55 ± 0.47

5.22 ± 0.28

2.83 ± 0.45

4.32 ± 0.57

5.03 ± 0.26

2PF Change (%, 750–850 nm/ 50 mV)

Table 13.1.  Linear and Non-linear Responses of Gen3 Dyes

27 ± 1

–

16 ± 1

109 ± 30